Chimeric HIV Env proteins comprising CD4 mini-proteins or CD4 mimetics that are capable of inducing neutralizing antibody responses against cryptic Env epitopes

ABSTRACT

Env-CD4 polypeptide complexes and hybrids that expose cryptic epitopes important in virus neutralization are disclosed. Method of diagnosis, treatment and prevention using the polypeptides are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. Ser. No.10/514,055, filed Nov. 8, 2004, pending, which is the U.S. NationalPhase of International Application No. PCT/US2003/014575, now expired,filed May 7, 2003, which claims priority to U.S. Provisional ApplicationNos. 60/459,314, filed Mar. 31, 2003, and 60/378,543, filed May 7, 2002,each of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was supported in whole, or in part, by NIAID-NIH HIVRADGrant No. 5P01 AI48225-03 from the National Institute of Allergy andInfectious Diseases. The Government has certain rights in the invention.

SEQUENCE LISTING

This application incorporates by reference the contents of a 23 kb textfile created on Aug. 11, 2010 and named “PAT051269_US_DIV.txt,” which isthe sequence listing for this application.

TECHNICAL FIELD

The invention relates generally to modified HIV envelope (Env)polypeptides that are useful as immunizing agents or for generating animmune response in a subject, for example a cellular immune response ora protective immune response. More particularly, the invention relatesEnv polypeptides such as monomeric or oligomeric gp120, gp140 or gp160complexed to CD4 proteins or mini-proteins wherein conserved, crypticepitopes participating in Env-CD4 and chemokine receptor binding areexposed. The invention also pertains to methods of using thesepolypeptides to elicit an immune response against a broad range of HIVsubtypes.

BACKGROUND OF THE INVENTION

The human immunodeficiency virus (HIV-1, also referred to as HTLV-III,LAV or HTLV-III/LAV) is the etiological agent of the acquired immunedeficiency syndrome (AIDS) and related disorders. (see, e.g.,Barre-Sinoussi, et al., (1983) Science 220:868-871; Gallo et al. (1984)Science 224:500-503; Levy et al., (1984) Science 225:840-842; Siegal etal., (1981) N Engl. J Med. 305:1439-1444). AIDS patients usually have along asymptomatic period followed by the progressive degeneration of theimmune system and the central nervous system. Replication of the virusis highly regulated, and both latent and lytic infection of the CD4positive helper subset of T-lymphocytes occurs in tissue culture (Zaguryet al., (1986) Science 231:850-853). Molecular studies of HIV-1 showthat it encodes a number of genes (Ratner et al., (1985) Nature313:277-284; Sanchez-Pescador et al., (1985) Science 227:484-492),including three structural genes—gag, pol and env—that are common to allretroviruses. Nucleotide sequences from viral genomes of otherretroviruses, particularly HIV-2 and simian immunodeficiency viruses,SIV (previously referred to as STLV-III), also contain these structuralgenes. (Guyader et al., (1987) Nature 326:662-669). The envelope proteinof HIV-1, HIV-2 and SIV is a glycoprotein of about 160 kd (gp160).During virus infection of the host cell, gp160 is cleaved by host cellproteases to form gp120 and the integral membrane protein, gp41. Thegp41 portion is anchored in the membrane bilayer of virion, while thegp120 segment protrudes into the surrounding environment. gp120 and gp41are more covalently associated and free gp120 can be released from thesurface of virions and infected cells.

Crystallography studies of the gp120 core polypeptide indicate that thispolypeptide is folded into two major domains having certain emanatingstructures. The inner domain (inner with respect to the N and Cterminus) features a two-helix, two-stranded bundle with a smallfive-stranded β-sandwich at its termini-proximal end and a projection atthe distal end from which the V1/V2 stem emanates. The outer domain is astaked double barrel that lies along side the inner domain so that theouter barrel and inner bundle axes are approximately parallel. Betweenthe distal inner domain and the distal outer domain is a four-strandedbridging sheet that holds a peculiar minidomain in contact with, butdistinct from, the inner, the outer domain, and the V1N2 domain. Thebridging sheet is composed of four β-strand structures (β-3, β-2, β-21,β-20). The bridging region is packed primarily over the inner domain,although some surface residues of the outer domain, such as Phe 382,reach into the bridging sheet to form part of its hydrophobic core. See,also WO 00/39303.

The basic unit of the β-sheet conformation of the bridging sheet regionis the β-strand that exists as a less tightly coiled helix, with 2.0residues per turn. The β-strand conformation is only stable whenincorporated into β-sheet, where hydrogen bonds with close to optimalgeometry are formed between the peptide groups on adjacent β-strands;the dipole moments of the strands are also aligned favorably. Sidechains from adjacent residues of the same strand protrude from oppositesides of the sheet and do not interact with each other, but havesignificant interactions with their backbone and with the side chains ofneighboring strands. For a general description of β-sheets, see, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); and A. L. Lehninger, Biochemistry (WorthPublishers, Inc., 1975).

The gp120 polypeptide is instrumental in mediating entry into the hostcell. Recent studies have indicated that binding of CD4 to gp120 inducesa conformational change in Env that allows for binding to a co-receptor(e.g., a chemokine receptor) and subsequent entry of the virus into thecell. (Wyatt, R., et al. (1998) Nature 393:705-711; Kwong, P., et al.(1998) Nature 393:648-659). It appears as though CD4 is bound into adepression formed at the interface of the outer domain, the inner domainand the bridging sheet of gp120.

Immunogenicity of the gp120 polypeptide has also been studied. Forexample, individuals infected by HIV-1 usually develop antibodies thatcan neutralize the virus in in vitro assays, and this response isdirected primarily against linear neutralizing determinants in the thirdvariable loop of gp120 glycoprotein (Javaherian, K., et al. (1989) Proc.Natl. Acad. Sci. 86:6786-6772; Matsushita, M., et al. (1988) J. Virol.62:2107-2144; Putney, S., et al. (1986) Science 234:1392-1395; Rushe, J.R., et al. (1988) Proc. Nat. Acad. Sci. USA 85: 3198-3202). However,these antibodies generally exhibit the ability to neutralize only alimited number of HIV-1 strains (Matthews, T. (1986) Proc. Natl. Acad.Sci. USA. 83:9709-9713; Nara, P. L., et al. (1988) J. Virol.62:2622-2628; Palker, T. J., et al. (1988) Proc. Natl. Acad. Sci. USA.85:1932-1936). Later in the course of HIV infection in humans,antibodies capable of neutralizing a wider range of HIV-1 isolatesappear (Barre-Sinoussi, F., et al. (1983) Science 220:868-871;Robert-Guroff, M., et al. (1985) Nature (London) 316:72-74; Weis, R., etal. (1985) Nature (London) 316:69-72; Weis, R., et al. (1986) Nature(London) 324:572-575).

Stamatatos et al (1998) AIDS Res Hum Retroviruses 14(13):1129-39 showthat a deletion of the variable region 2 from a HIV-1_(SF162) virus,which utilizes the CCR-5 co-receptor for virus entry, rendered the virushighly susceptible to serum-mediated neutralization. This V2 deletedvirus was also neutralized by sera obtained from patients infected notonly with Glade B HIV-1 isolates but also with Glade A, C, D and F HIV-1isolates. However, deletion of the variable region 1 had no effect.Deletion of the variable regions 1 and 2 from a LAI isolate HIV-I_(IIIB)also increased the susceptibility to neutralization by monoclonalantibodies whose epitopes are located within the V3 loop, theCD4-binding site, and conserved gp120 regions (Wyatt, R., et al. (1995)J. Virol. 69:5723-5733). Rabbit immunogenicity studies done with theHIV-1 virus with deletions in the V1/V2 and V3 region from the LAIstrain, which uses the CXCR4 co-receptor for virus entry, showed noimprovement in the ability of Env to raise neutralizing antibodies (Leuet al. (1998) AIDS Res. and Human Retroviruses. 14:151-155).

Further, a subset of the broadly reactive antibodies, found in mostinfected individuals, interferes with the binding of gp120 and CD4(Kang, C.-Y., et al. (1991) Proc. Natl. Acad. Sci. USA. 88:6171-6175;McDougal, J. S., et al. (1986) J. Immunol. 137:2937-2944). Otherantibodies are believed to bind to the chemokine receptor-binding regionafter CD4 has bound to Env (Thali et al. (1993) J. Virol. 67:3978-3988).The fact that neutralizing antibodies generated during the course of HIVinfection do not provide permanent antiviral effect may in part be dueto the generation of “neutralization escapes” virus mutants and to thegeneral decline in the host immune system associated with pathogenesis.In contrast, the presence of pre-existing neutralizing antibodies uponinitial HIV-1 exposure will likely have a protective effect.

It is widely thought that a successful vaccine should be able to inducea strong, broadly neutralizing antibody response against diverse HIV-1strains (Montefiori and Evans (1999) AIDS Res. Hum. Ret. 15(8):689-698;Bolognesi, D. P., et al. (1994) Ann. Int. Med. 8:603-611; Haynes, B.,F., et al. (1996) Science; 271: 324-328.). Neutralizing antibodies, byattaching to the incoming virions, can reduce or even prevent theirinfectivity for target cells and prevent the cell-to-cell spread ofvirus in tissue culture (Hu et al. (1992) Science 255:456-459; Burton,D. R. and Montefiori, D. (1997) AIDS 11(suppl. A): 587-598). However asdescribed above, antibodies directed against gp120 do not generallyexhibit broad antibody responses against different HIV strains.

Currently, the focus of vaccine development, from the perspective ofhumoral immunity, is on the neutralization of primary isolates thatutilize the CCR5 chemokine co-receptor believed to be important in virusentry (Zhu, T., et al. (1993) Science 261:1179-1181; Fiore, J., et al.(1994) Virology; 204:297-303). These viruses are generally much moreresistant to antibody neutralization than T-cell line adapted strainsthat use the CXCR4 co-receptor, although both can be neutralized invitro by certain broadly and potent acting monoclonal antibodies, suchas IgG1b12, 2G12 and 2F5 (Trkola, A., et al. (1995) J. Virol.69:6609-6617; D'Sousa P M., et al (1997) J. Infect. Dis. 175:1062-1075).These monoclonal antibodies are directed to the CD4 binding site, aglycosylation site and to the gp41 fusion domain, respectively.

The problem that remains, however, is that it is not known how to induceantibodies of the appropriate specificity by vaccination. Antibodies(Abs) elicited by gp120 glycoprotein from a given isolate are usuallyonly able to neutralize closely related viruses generally from similar,usually from the same, HIV-1 subtype. Thus, there remains a need for Envantigens that can elicit an immunological response (e.g., neutralizingand/or protective antibodies) in a subject against multiple HIV strainsand subtypes, for example when administered as a vaccine.

SUMMARY OF THE INVENTION

The present invention solves these and other problems by providinghybrid Env-CD4 proteins and Env polypeptides (e.g., native or modifiedgp120) complexed to novel, CD4 mini-proteins or mimics (mimetics) inorder to expose epitopes in or near the CD4 binding site.

In one aspect, the invention includes a polynucleotide encoding a hybridHIV Env-CD4 protein, where the protein include amino acid sequences froman HIV Env and CD4 amino acid sequences. In certain embodiments, theCD4-encoding polynucleotide sequences are inserted into (or embedded)within the HIV Env-encoding polynucleotides sequences, for exampleinserted in place of polynucleotides encoding for one or more amino acidresidues in the variable regions (V1-V5) of HIV Env. Thus, the inventionincludes a polynucleotide comprising a first sequence encoding an HIVEnv polypeptide and a second sequence encoding a CD4 protein, whereinthe second sequence is inserted into, and in proper reading frame with,the first sequence. In certain embodiments, the HIV Env polypeptide ofthe hybrid is encoded by a modified gp120 sequence (SEQ ID NO:5) and thesecond sequence comprises one or more of SEQ ID NO:1-4. In certainembodiments, the HIV Env polypeptide is based on strain SF162. In any ofthe polypeptides described herein, one or more of the Env variableregions (V1-V5) regions may be modified (e.g., contain deletions and/orsubstitutions).

In any of the polynucleotides described herein can further comprise oneor more linker sequences, for example, linker sequences flanking theCD4-encoding sequence (second sequence). Further, when any of thepolynucleotides described herein are expressed, the CD4 peptide ispreferably bound to (complexed) to the HIV Env polypeptide such thatcryptic epitopes are exposed in the modified Env polypeptide.

In yet another aspect, the invention includes polypeptides encoding byany of the polynucleotides described herein. Thus, in certainembodiments, the polypeptide comprises a hybrid Env-CD4 protein, forexample, a hybrid polypeptide comprising an Env polypeptide (e.g.,native or modified gp160, gp140, oligomeric-gp140, gp120) and a CD4protein (e.g., sCD4, CD4 mimetics, CD4 mini-proteins such as SEQ IDNO:1-4, etc.). The Env polypeptide may include one or moremodifications, for example deletions in one or more of the variableregions. In certain embodiments, the CD4 protein is inserted into theEnv polypeptide, for example into one of the deletions.

In yet another aspect, the invention includes polypeptide complexescomprising an HIV Env polypeptide (e.g., native or modified gp160,gp140, oligomeric-gp140, gp120) complexed to a CD4 protein (e.g., sCD4,CD4 mimetics, CD4 mini-proteins such as SEQ ID NO:1-4, etc.). The HIVEnv polypeptide and CD4 protein can be complexed by crosslinking (e.g.,using formaldehyde); using a fixative (e.g., formalin); and/or cancomplex spontaneously to form a covalent bond under suitable conditions.Polynucleotides encoding the components of the complex are alsodescribed herein, for example one or more polynucleotide encoding boththe Env and CD4 polypeptides (e.g., soluble CD4) can be expressed andthe resulting proteins complexed together.

In yet another aspect, the invention includes immunogenic compositionscomprising any of the polynucleotides and/or polypeptides describedherein. In certain embodiments, the immunogenic compositions furthercomprise one or more adjuvants.

In a still further aspect, the invention includes a cell comprising anyof the polynucleotides and/or polypeptides described herein. Thepolynucleotide sequences are preferably operably linked to controlelements compatible with expression in the selected cell. The cell canbe, for example, a mammalian cell. (e.g., BHK, VERO, HT1080, 293, RD,COS-7, and CHO cells); an insect cell (e.g., Trichoplusia ni (Tn5) orSf9 cells); a bacterial cell; a yeast cell; a plant cell; an antigenpresenting cell; a lymphoid cell selected from the group consisting ofmacrophage, monocytes, dendritic cells, B-cells, T-cells, stem cells,and progenitor cells thereof; a primary cell; an immortalized cell;and/or a tumor-derived cell.

In another aspects, the invention includes a gene delivery vector foruse in a mammalian subject, comprising a suitable gene delivery vectorfor use in said subject, wherein the vector comprises any of thepolynucleotides described herein operably linked to control elementscompatible with expression in the subject.

In yet another aspect, the invention includes a method of producingantibodies that bind to cryptic epitopes of HIV Env. In certainembodiments, the methods comprising the step of administering any of thepolypeptides described herein to a subject under conditions that allowproduction of antibodies. In other embodiments, epitopes involved inneutralization of HIV are identified, for example by identifying anepitope (e.g., determining the sequence of epitope) and antibodiesproduced by administering the identified epitope(s) to a subject underconditions that allow production of antibodies. The invention alsoincludes antibodies produced by any of the methods described herein. Inany of the methods and compositions described herein, the antibodies canbe neutralizing antibodies, monoclonal antibodies and/or polyclonalantibodies). In certain embodiments, the antibodies produced in thesubject are then isolated.

In a still further aspect, the invention includes a method for producinga hybrid Env-CD4 polypeptide comprising incubating any of the cellsdescribed herein, under conditions suitable for producing saidpolypeptide

In yet another aspect, the invention includes a method of inducing animmune response (e.g., a humoral response such as a neutralizingantibody response and/or a cellular immune response) in subjectcomprising, administering any of the polynucleotides, polypeptidesand/or immunogenic compositions described herein to a subject in anamount sufficient to induce an immune response in the subject. Incertain embodiments, the method comprises transfecting cells ex vivo andreintroducing the transfected cells into the subject. In otherembodiments, the method includes DNA immunization of a subject, forexample, by introducing any of the polynucleotides and/or gene deliveryvectors described herein into said subject under conditions that arecompatible with expression of said expression cassette and production ofa polypeptide in said subject. In other embodiments, the methodscomprise (a) administering a first composition comprising any of thepolynucleotides described herein in a priming step and (b) administeringa second composition comprising any of the polypeptides describedherein, as a booster, in an amount sufficient to induce an immuneresponse in the subject. In any of the methods described herein, thevectors may comprise non-viral vectors or viral vectors such asretroviral (e.g., lentiviral) vectors. Further, the polynucleotidesand/or vector may be introduced, for example, using a particulatecarrier (e.g., coated on a gold or tungsten particle and said coatedparticle is delivered to said subject using a gene gun) or encapsulatedin a liposome preparation. In any of the methods described herein, thesubject can be a mammal, for example a human or non-human mammal and theintroduction can be intramuscularly, intramucosally, intranasally,subcutaneously, intradermally, transdermally, intravaginally,intrarectally, orally and/or intravenously.

These and other embodiments of the subject invention will readily occurto those of skill in the art in light of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO:6) depicts the primary amino acid sequence of the Envpolypeptide precursor of HIV-1_(SF2) (hereinafter “SF2”) strain (See,also GenBank Accession No. VCLJA2 and Sanchez-Pescador et al. (1984)Science 227:484).

FIG. 2 (SEQ ID NO:7) depicts amino acid sequence alignment of CDR2-likeloop of human CD4, scorpion scyllatoxin, engineered CD4 mimetic (CD4M3),double (CD4M8) and quintuple (CD4M9) mutants.

FIG. 3, panels a and b, are graphs depicting immunogenicity ofgp120-sCD4 complexes. Panel a depicts antibody responses to gp120induced by fixed (formalin or formaldehyde) and unfixed gp120-sCD4 atdifferent time points. The black bars show fixed complexes and the graybars show unfixed complexes. Panel b shows antibody responses to CD4(pooled specimens).

FIG. 4, panels A and B, depict characterization of gp120-CD4 complexesusing size exclusion chromatography (panel A) and SDS-PAGE (panel B).The profiles and retention times of gp120, CD4 alone and as a complex asobtained using a size exclusion column (Panel A). SDS-PAGE analysis ofCD4 (lane 1), gp120 SF2 (lane 3) and gp120-CD4 complex (lane 2).Molecular weight markers are shown in lane-4.

FIG. 5 is a bar graph depicting reactivities against gp120 and of CD4affinity purified antibodies. The left bar of each set shows anti-gp120reactivity and the right bar of each set shows anti-CD4 reactivity.

FIG. 6 is bar graph depicting primary isolate neutralizing antibodyactivity of gp120 column fractions. The left bar of each set showsactivity at 1:10 dilution; the center bar shows activity at 1:40dilution and the right bar shows activity at 1:160 dilution.

FIG. 7 (SEQ ID NO:5) depicts the amino acid sequence of an HIV gp120polypeptide encoded by a modified gp120-encoding polynucleotide sequence(gp120.modSF62). The V1 Loop is underlined; the V2 Loop is shown inbold; the V3 Loop is shown in italics; the V4 Loop is shown in dashedunderlining; and the V5 Loop is shown in double underlining.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, viralimmunobiology, molecular biology and recombinant DNA techniques withinthe skill of the art. Such techniques are explained fully in theliterature. See, e.g., T. E. Creighton, Proteins: Structures andMolecular Properties (W.H. Freeman and Company, 1993); Nelson L. M. andJerome H. K. HIV Protocols in Methods in Molecular Medicine, vol. 17,1999; Sambrook, et al., Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory, 1989); F. M. Ausubel et al. Current Protocolsin Molecular Biology, Greene Publishing Associates & Wiley InterscienceNew York; and Lipkowitz and Boyd, Reviews in Computational Chemistry,volumes 1-present (Wiley-VCH, New York, N.Y., 1999).

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a polypeptide” includes a mixture of two or morepolypeptides, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polypeptide,” and “protein” are used interchangeably hereinto denote any polymer of amino acid residues. The terms encompasspeptides, oligopeptides, dimers, multimers, and the like. Suchpolypeptides can be derived from natural sources or can be synthesizedor recombinantly produced. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation, etc.

A polypeptide as defined herein is generally made up of the 20 naturalamino acids Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (O), Glu(E), Gly (G), H is (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro(P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V) and may also includeany of the several known amino acid analogs, both naturally occurringand synthesized analogs, such as but not limited to homoisoleucine,asaleucine, 2-(methylenecyclopropyl)glycine, S-methylcysteine,S-(prop-1-enyl)cysteine, homoserine, ornithine, norleucine, norvaline,homoarginine, 3-(3-carboxyphenyl)alanine, cyclohexylalanine, mimosine,pipecolic acid, 4-methylglutamic acid, canavanine, 2,3-diaminopropionicacid, and the like. Further examples of polypeptide agents that willfind use in the present invention are set forth below.

By “geometry” or “tertiary structure” of a polypeptide or protein ismeant the overall 3-D configuration of the protein. As described herein,the geometry can be determined, for example, by crystallography studiesor by using various programs or algorithms that predict the geometrybased on interactions between the amino acids making up the primary andsecondary structures.

By “wild type” polypeptide, polypeptide agent or polypeptide drug, ismeant a naturally occurring polypeptide sequence, and its correspondingsecondary structure. An “isolated” or “purified” protein or polypeptideis a protein that is separate and discrete from a whole organism withwhich the protein is normally associated in nature. It is apparent thatthe term denotes proteins of various levels of purity. Typically, acomposition containing a purified protein will be one in which at leastabout 35%, preferably at least about 40-50%, more preferably, at leastabout 75-85%, and most preferably at least about 90% or more, of thetotal protein in the composition will be the protein in question.

The terms “CD4 mini-protein” and “mini CD4 protein” are usedinterchangeably to refer to any polypeptide that interacts with Env(e.g., gp120), preferably such that epitopes (e.g., cryptic epitopes) inor near the CD4 and/or chemokine receptor binding sites(s) are exposed.Thus, a CD mini-protein can be a less than full-length fragment of CD4.In addition, the term encompasses functional and structural homologs ofCD4 fragments, i.e., polypeptides that expose the cryptic epitopes on anEnv protein.

By “Env polypeptide” is meant a molecule derived from an envelopeprotein, preferably from HIV Env. The envelope protein of HIV-1 is aglycoprotein of about 160 kd (gp160). During virus infection of the hostcell, gp160 is cleaved by host cell proteases to form gp120 and theintegral membrane protein, gp41. The gp41 portion is anchored in (andspans) the membrane bilayer of virion, while the gp120 segment protrudesinto the surrounding environment. As there is no covalent attachmentbetween gp120 and gp41, free gp120 is released from the surface ofvirions and infected cells. Env polypeptides may also include gp140polypeptides. Env polypeptides can exist as monomers, dimers ormultimers.

By a “gp120 polypeptide” is meant a molecule derived from a gp120 regionof the Env polypeptide. Preferably, the gp120 polypeptide is derivedfrom HIV Env. The primary amino acid sequence of gp120 is approximately511 amino acids, with a polypeptide core of about 60,000 Daltons. Thepolypeptide is extensively modified by N-linked glycosylation toincrease the apparent molecular weight of the molecule to 120,000Daltons. The amino acid sequence of gp120 contains five relativelyconserved domains interspersed with five hypervariable domains. Thepositions of the 18 cysteine residues in the gp120 primary sequence ofthe HIV-1_(HXB-2) (hereinafter “HXB-2”) strain, and the positions of 13of the approximately 24 N-linked glycosylation sites in the gp120sequence are common to most, if not all, gp120 sequences. Thehypervariable domains contain extensive amino acid substitutions,insertions and deletions. Despite this variation, most, if not all,gp120 sequences preserve the virus's ability to bind to the viralreceptor CD4. A “gp120 polypeptide” includes both single subunits and/ormultimers.

Env polypeptides (e.g., gp120, gp140 and gp160) include a “bridgingsheet” comprised of 4 anti-parallel β-strands (β-2, β-3, β-20 and β-21)that form a β-sheet. Extruding from one pair of the β-strands (β-2 andβ-3) are two loops, V1 and V2. The β-2 sheet occurs at approximatelyamino acid residue 119 (Cys) to amino acid residue 123 (Thr) while β-3occurs at approximately amino acid residue 199 (Ser) to amino acidresidue 201 (Ile), relative to HXB-2. The “V1/V2 region” occurs atapproximately amino acid positions 126 (Cys) to residue 196 (Cys),relative to HXB-2. (see, e.g., Wyatt et al. (1995) J. Virol.69:5723-5733; Stamatatos et al. (1998) J. Virol. 72:7840-7845).Extruding from the second pair of β-strands (β-20 and β-21) is a“small-loop” structure, also referred to herein as “the bridging sheetsmall loop.” In HXB-2, β-20 extends from about amino acid residue 422(Gln) to amino acid residue 426 (Met) while β-21 extends from aboutamino acid residue 430 (Val) to amino acid residue 435 (Tyr). In variantSF162, the Met-426 is an Arg (R) residue. The “small loop” extends fromabout amino acid residue 427 (Trp) through 429 (Lys), relative to HXB-2.Alignment of the amino acid sequences of Env polypeptide gp160 of anyHIV variant can be determined relative to other variants, such as HXB-2,as described for example, in WO 00/39303.

Furthermore, an “Env polypeptide” or “gp120 polypeptide” as definedherein is not limited to a polypeptide having the exact sequencedescribed herein. Indeed, the HIV genome is in a state of constant fluxand contains several variable domains that exhibit relatively highdegrees of variability between isolates. It is readily apparent that theterms encompass Env (e.g., gp120) polypeptides from any of theidentified HIV isolates, as well as newly identified isolates, andsubtypes of these isolates. Descriptions of structural features aregiven herein with reference to HXB-2. One of ordinary skill in the artin view of the teachings of the present disclosure and the art candetermine corresponding regions in other HIV variants (e.g., isolatesHIV_(IIIb), HIV_(SF2), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV),HIV_(LAI), HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strainsfrom diverse subtypes (e.g., subtypes, A through G, and O), HIV-2strains and diverse subtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)), andsimian immunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition(W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fieldsand D. M. Knipe, eds. 1991); Virology, 3rd Edition (Fields, B N, D MKnipe, P M Howley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.;for a description of these and other related viruses), using forexample, sequence comparison programs (e.g., BLAST and others describedherein) or identification and alignment of structural features (e.g., aprogram such as the “ALB” program described herein that can identifyβ-sheet regions). The actual amino acid sequences of the modified Envpolypeptides can be based on any HIV variant.

Additionally, the term “Env polypeptide” (e.g., “gp120 polypeptide”)encompasses proteins that include additional modifications to the nativesequence, such as additional internal deletions, additions andsubstitutions. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughnaturally occurring mutational events. Thus, for example, if the Envpolypeptide is to be used in vaccine compositions, the modificationsmust be such that immunological activity (i.e., the ability to elicit anantibody response to the polypeptide) is not lost. Similarly, if thepolypeptides are to be used for diagnostic purposes, such capabilitymust be retained.

Thus, a “modified Env polypeptide” is an Env polypeptide (e.g., gp120 asdefined above), which has been complexed to a CD4 mini protein and,optionally, modified in the variable regions V1 and V2. The Envpolypeptide may be monomeric or oligomeric. Generally, complexed Env(e.g., gp120) polypeptides result in exposure of epitopes in or near theCD4 binding site, while allowing correct folding (e.g., correctgeometry) of the Env polypeptide. Additionally, modifications (e.g.,truncations) to the variable loop regions (V1, V2, V3, V4 and/or V5) mayalso be made. Although not all possible variable region modificationshave been exemplified herein, it is to be understood that otherdisrupting modifications are also encompassed by the present invention.

The term “hybrid” protein, when used in reference to Env-CD4 hybridproteins, refers to polypeptides in which CD4 amino acid sequences(e.g., mini proteins, mimetics, etc.) are inserted into (flanked by) Envamino acid sequences. Non-limiting examples of Env-CD4 hybrids aredescribed in Example 4 in which various CD4 mini protein sequences areinserted in place of (substituted for) one or more of the variable loopregions (V1-V5) of an Env protein.

Normally, hybrid or modified polypeptides as described herein capable ofsecretion into growth medium in which an organism expressing the proteinis cultured. However, for purposes of the present invention, suchpolypeptides may also be recovered intracellularly. Secretion intogrowth media is readily determined using a number of detectiontechniques, including, e.g., polyacrylamide gel electrophoresis and thelike, and immunological techniques such as Western blotting andimmunoprecipitation assays as described in, e.g., InternationalPublication No. WO 96/04301.

A polypeptide is produced “intracellularly” when it is found within thecell, either associated with components of the cell, such as inassociation with the endoplasmic reticulum (ER) or the Golgi Apparatus,or when it is present in the soluble cellular fraction. The Env, CD4 andhybrid polypeptides of the present invention may also be secreted intogrowth medium so long as sufficient amounts of the polypeptides remainpresent within the cell such that they can be purified from cell lysatesusing techniques described herein.

An “immunogenic” protein is a molecule that includes at least oneepitope such that the molecule is capable of either eliciting animmunological reaction in an individual to which the protein isadministered or, in the diagnostic context, is capable of reacting withantibodies directed against the HIV in question.

By “epitope” is meant a site on an antigen to which specific B cellsand/or T cells respond, rendering the molecule including such an epitopecapable of eliciting an immunological reaction or capable of reactingwith HIV antibodies present in a biological sample. The term is alsoused interchangeably with “antigenic determinant” or “antigenicdeterminant site.” An epitope can comprise 3 or more amino acids in aspatial conformation unique to the epitope. Generally, an epitopeconsists of at least 5 such amino acids and, more usually, consists ofat least 8-10 such amino acids. Methods of determining spatialconformation of amino acids are known in the art and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance. Furthermore, the identification of epitopes in a givenprotein is readily accomplished using techniques well known in the art,such as by the use of hydrophobicity studies and by site-directedserology. See, also, Geysen et al., Proc. Natl. Acad. Sci. USA (1984)81:3998-4002 (general method of rapidly synthesizing peptides todetermine the location of immunogenic epitopes in a given antigen); U.S.Pat. No. 4,708,871 (procedures for identifying and chemicallysynthesizing epitopes of antigens); and Geysen et al., MolecularImmunology (1986) 23:709-715 (technique for identifying peptides withhigh affinity for a given antibody). Antibodies that recognize the sameepitope can be identified in a simple immunoassay showing the ability ofone antibody to block the binding of another antibody to a targetantigen. A “cryptic epitope” refers generally to an epitope that isexposed only in certain conformations of the protein.

An “immunological response” or “immune response” as used herein is thedevelopment in the subject of a humoral and/or a cellular immuneresponse to the Env (e.g., gp120) polypeptide when the polypeptide ispresent in a vaccine composition. These antibodies may also neutralizeinfectivity, and/or mediate antibody-complement or antibody dependentcell cytotoxicity to provide protection to an immunized host.Immunological reactivity may be determined in standard immunoassays,such as a competition assays, well known in the art.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA of interest into a host cell. Such methods canresult in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from alphaviruses, pox virusesand vaccinia viruses. When used for immunization, such gene deliveryexpression vectors may be referred to as vaccines or vaccine vectors.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as, the following:(i) hybrid (chimeric) antibody molecules (see, for example, Winter etal. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); (ii)F(ab′)2 and F(ab) fragments; (iii) Fv molecules (noncovalentheterodimers, see, for example, Inbar et al. (1972) Proc. Natl. Acad.Sci. USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096);(iv) single-chain Fv molecules (sFv) (see, for example, Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (v) dimeric andtrimeric antibody fragment constructs; (vi) humanized antibody molecules(see, for example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyanet al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB2,276,169, published 21 Sep. 1994); (vii) Mini-antibodies or minibodies(i.e., sFv polypeptide chains that include oligomerization domains attheir C-termini, separated from the sFv by a hinge region; see, e.g.,Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J.Immunology 149B:120-126); and, (vii) any functional fragments obtainedfrom such molecules, wherein such fragments retain specific-bindingproperties of the parent antibody molecule.

Thus, the term “antibody” refers to a polypeptide or group ofpolypeptides that comprise at least one antigen binding site. An“antigen binding site” is formed from the folding of the variabledomains of an antibody molecule(s) to form three-dimensional bindingsites with an internal surface shape and charge distributioncomplementary to the features of an epitope of an antigen, which allowsspecific binding to form an antibody-antigen complex. An antigen bindingsite may be formed from a heavy- and/or light-chain domain (VH and VL,respectively), which form hypervariable loops that contribute to antigenbinding. The term “antibody” includes, without limitation, polyclonalantibodies, monoclonal antibodies, chimeric antibodies, alteredantibodies, univalent antibodies, Fab proteins, and single-domainantibodies. In many cases, the binding phenomena of antibodies toantigens is equivalent to other ligand/anti-ligand binding.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptideepitope(s). Serum from the immunized animal is collected and treatedaccording to known procedures. If serum containing polyclonal antibodiesto Env-CD4 complexes and/or hybrids contain antibodies to otherantigens, the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art, see for example, Mayer and Walker, eds.(1987) IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (AcademicPress, London).

One skilled in the art can also readily produce monoclonal antibodiesdirected against epitopes exposed from Env-CD4 complexes and hybrids.The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal antibody-producing cell lines can be created bycell fusion, and also by other techniques such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al. (1980) HYBRIDOMA TECHNIQUES;Hammerling et al. (1981), MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS;Kennett et al. (1980) MONOCLONAL ANTIBODIES; see also, U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500;4,491,632; and 4,493,890. Panels of monoclonal antibodies can bescreened for various properties; i.e., for isotype, epitope affinity,etc. As used herein, a “single domain antibody” (dAb) is an antibodythat is comprised of an HL domain, which binds specifically with adesignated antigen. A dAb does not contain a VL domain, but may containother antigen binding domains known to exist to antibodies, for example,the kappa and lambda domains. Methods for preparing dabs are known inthe art. See, for example, Ward et al, Nature 341: 544 (1989).

Antibodies can also be comprised of VH and VL domains, as well as otherknown antigen binding domains. Examples of these types of antibodies andmethods for their preparation and known in the art (see, e.g., U.S. Pat.No. 4,816,467, which is incorporated herein by reference), and includethe following. For example, “vertebrate antibodies” refers to antibodiesthat are tetramers or aggregates thereof, comprising light and heavychains which are usually aggregated in a “Y” configuration and which mayor may not have covalent linkages between the chains. In vertebrateantibodies, the amino acid sequences of the chains are homologous withthose sequences found in antibodies produced in vertebrates, whether insitu or in vitro (for example, in hybridomas). Vertebrate antibodiesinclude, for example, purified polyclonal antibodies and monoclonalantibodies, methods for the preparation of which are described infra.

“Hybrid antibodies” are antibodies where chains are separatelyhomologous with reference to mammalian antibody chains and representnovel assemblies of them, so that two different antigens areprecipitable by the tetramer or aggregate. In hybrid antibodies, onepair of heavy and light chains are homologous to those found in anantibody raised against a first antigen, while a second pair of chainsare homologous to those found in an antibody raised against a secondantibody. This results in the property of “divalence”, i.e., the abilityto bind two antigens simultaneously. Such hybrids can also be formedusing chimeric chains, as set forth below.

“Chimeric antibodies” refers to antibodies in which the heavy and/orlight chains are fusion proteins. Typically, one portion of the aminoacid sequences of the chain is homologous to corresponding sequences inan antibody derived from a particular species or a particular class,while the remaining segment of the chain is homologous to the sequencesderived from another species and/or class. Usually, the variable regionof both light and heavy chains mimics the variable regions or antibodiesderived from one species of vertebrates, while the constant portions arehomologous to the sequences in the antibodies derived from anotherspecies of vertebrates. However, the definition is not limited to thisparticular example. Also included is any antibody in which either orboth of the heavy or light chains are composed of combinations ofsequences mimicking the sequences in antibodies of different sources,whether these sources be from differing classes or different species oforigin, and whether or not the fusion point is at the variable/constantboundary. Thus, it is possible to produce antibodies in which neitherthe constant nor the variable region mimic know antibody sequences. Itthen becomes possible, for example, to construct antibodies whosevariable region has a higher specific affinity for a particular antigen,or whose constant region can elicit enhanced complement fixation, or tomake other improvements in properties possessed by a particular constantregion.

Another example is “altered antibodies”, which refers to antibodies inwhich the naturally occurring amino acid sequence in a vertebrateantibody has been varies. Utilizing recombinant DNA techniques,antibodies can be redesigned to obtain desired characteristics. Thepossible variations are many, and range from the changing of one or moreamino acids to the complete redesign of a region, for example, theconstant region. Changes in the constant region, in general, to attaindesired cellular process characteristics, e.g., changes in complementfixation, interaction with membranes, and other effector functions.Changes in the variable region can be made to alter antigen bindingcharacteristics. The antibody can also be engineered to aid the specificdelivery of a molecule or substance to a specific cell or tissue site.The desired alterations can be made by known techniques in molecularbiology, e.g., recombinant techniques, site-directed mutagenesis, etc.

Yet another example are “univalent antibodies”, which are aggregatescomprised of a heavy-chain/light-chain dimer bound to the Fc (i.e.,stem) region of a second heavy chain. This type of antibody escapesantigenic modulation. See, e.g., Glennie et al. Nature 295: 712 (1982).Included also within the definition of antibodies are “Fab” fragments ofantibodies. The “Fab” region refers to those portions of the heavy andlight chains which are roughly equivalent, or analogous, to thesequences which comprise the branch portion of the heavy and lightchains, and which have been shown to exhibit immunological binding to aspecified antigen, but which lack the effector Fc portion. “Fab”includes aggregates of one heavy and one light chain (commonly known asFab′), as well as tetramers containing the 2H and 2L chains (referred toas F(ab)₂), which are capable of selectively reacting with a designatedantigen or antigen family. Fab antibodies can be divided into subsetsanalogous to those described above, i.e., “vertebrate Fab”, “hybridFab”, “chimeric Fab”, and “altered Fab”. Methods of producing Fabfragments of antibodies are known within the art and include, forexample, proteolysis, and synthesis by recombinant techniques.

“Antigen-antibody complex” refers to the complex formed by an antibodythat is specifically bound to an epitope on an antigen.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An implementation of this algorithm for nucleic acid and peptidesequences is provided by the Accelrys in their BestFit utilityapplication. The default parameters for this method are described in thesupplier's (Accelrys) materials. Other equally suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art.

For example, percent identity of a particular nucleotide sequence to areference sequence can be determined using the homology algorithm ofSmith and Waterman with a default scoring table and a gap penalty of sixnucleotide positions. Another method of establishing percent identity inthe context of the present invention is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages, theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated,the “Match” value reflects “sequence identity.” Other suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art, such as the alignment program BLAST, whichcan also be used with default parameters. For example, BLASTN and BLASTPcan be used with the following default parameters: geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://www.ncbi.nlm.gov/cgi-bin/BLAST.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence in the above programs. Forexample, the search parameters may vary based on the size of thesequence in question. Thus, for example, a representative embodiment ofthe present invention would include an isolated polynucleotide orpolypeptide having X contiguous nucleotides or amino acids, wherein (i)the X contiguous nucleotides or amino acids have at least about 50%identity to Y contiguous nucleotides or amino acids derived from any ofthe sequences described herein, (ii) X equals Y, and (iii) X is greaterthan or equal to 6 nucleotides and up to 5000 nucleotides or 3 aminoacids to 500 amino acids, preferably greater than or equal to 8nucleotides and up to 5000 nucleotides or 15 amino acids to 500 aminoacids, more preferably 10-12 nucleotides and up to 5000 nucleotides or20 amino acids to 500 amino acids, including all integer values fallingwithin the above-described ranges.

Computer programs are also available to determine the likelihood ofcertain polypeptides to form structures such as β-sheets. One suchprogram, described herein, is the “ALB” program for protein andpolypeptide secondary structure calculation and predication. Inaddition, secondary protein structure can be predicted from the primaryamino acid sequence, for example using protein crystal structure andaligning the protein sequence related to the crystal structure (e.g.,using Molecular Operating Environment (MOE) programs available from theChemical Computing Group Inc., Montreal, P.Q., Canada). Other methods ofpredicting secondary structures are described, for example, in Garnieret al. (1996) Methods Enzymol. 266:540-553; Geourjon et al. (1995)Comput. Applic. Biosci. 11:681-684; Levin (1997) Protein Eng.10:771-776; and Rost et al. (1993) J. Molec. Biol. 232:584-599.

Homology can also be determined by hybridization of polynucleotidesunder conditions that form stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two DNA, or twopolypeptide sequences are “substantially homologous” to each other whenthe sequences exhibit at least about 80%-85%, preferably at least about90%, and most preferably at least about 95%-98% sequence identity over adefined length of the molecules, as determined using the methods above.As used herein, substantially homologous also refers to sequencesshowing complete identity to the specified DNA or polypeptide sequence.DNA sequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriate:hybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

A “coding sequence” or a sequence that “encodes” a selected protein, isa nucleic acid sequence that is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to cDNA fromviral nucleotide sequences as well as synthetic and semisynthetic DNAsequences and sequences including base analogs. A transcriptiontermination sequence may be located 3′ to the coding sequence.

“Control elements” refers collectively to promoter sequences, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, whichcollectively provide for the transcription and translation of a codingsequence in a host cell. Not all of these control elements need alwaysbe present so long as the desired gene is capable of being transcribedand translated.

A control element “directs the transcription” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence when RNApolymerase is present. The control elements need not be contiguous withthe coding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between, e.g., a promoter sequence and thecoding sequence and the promoter sequence can still be considered“operably linked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting prokaryotic microorganisms or eukaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

By “vertebrate subject” is meant any member of the subphylum chordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from an individual, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, samplesderived from the gastric epithelium and gastric mucosa, tears, saliva,milk, blood cells, organs, biopsies and also samples of in vitro cellculture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

The terms “label” and “detectable label” refer to a molecule capable ofdetection, including, but not limited to, radioactive isotopes,fluorescers, chemiluminescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, chromophores, dyes, metal ions, metalsols, ligands (e.g., biotin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof that is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels that may be used with the invention include, but are notlimited to fluorescein, rhodamine, dansyl, umbelliferone, Texas red,luminol, acradimum esters, NADPH, beta-galactosidase, horseradishperoxidase, glucose oxidase, alkaline phosphatase and urease.

Overview

The present invention concerns hybrid Env-CD4 polypeptide and Env-CD4complexes (and polynucleotides encoding the hybrids and/or complexes) aswell as the use of these molecules. Without being bound by a particulartheory, it appears that it has been difficult to generate immunologicalresponses against Env because the CD4 binding site (and/or the CCRbinding site) is buried between the outer domain, the inner domain andthe V1/V2 domains of Env. Thus, although deletion of the V1/V2 domainmay render the virus more susceptible to neutralization by monoclonalantibody directed to the CD4 site, the conformation of Env prior to CD4binding may prevent an antibody response. Thus, the present inventionprovides Env polypeptides complexed to CD4 proteins (e.g., soluble or CDmini-proteins) or hybrid Env-CD4 polypeptides. Binding of CD4 to Env inthese molecules causes a conformational change in Env that exposes oneor more epitopes (e.g., cryptic epitopes) in or near the CD4 bindingsite, which in turn allows the generation of an immune response (e.g., aneutralizing antibody response) to Env.

Various forms of the different embodiments of the invention, describedherein, may be combined.

Env Polypeptides

The Env polypeptide portion of the complexes described herein can bederived from an envelope protein, preferably from HIV Env. As notedabove, the envelope protein of HIV-1 is a glycoprotein of about 160 kd(gp160). During virus infection of the host cell, gp160 is cleaved byhost cell proteases to form gp120 and the integral membrane protein,gp41. The gp41 portion is anchored in (and spans) the membrane bilayerof virion, while the gp120 segment protrudes into the surroundingenvironment. As there is no covalent attachment between gp120 and gp41,free gp120 is released from the surface of virions and infected cells.Env polypeptides may also include gp140 polypeptides.

In certain embodiments, the Env polypeptide component of the complex isa monomer or a dimer. In preferred embodiments, the Env polypeptidecomponent is an oligomeric Env polypeptide. The primary sequence of theEnv polypeptide precursor of HIV-1_(SF2) (hereinafter “SF2”) strain isshown in FIG. 1. The gp120 amino acid sequence (including leadersequence) extends from amino acids 1-509 of FIG. 1). The polypeptidecontains approximately 24 N-linked glycosylation sites that are commonto most, if not all, gp120 sequences. As suggested by their name, thehypervariable domains contain extensive amino acid substitutions,insertions and deletions as between strains. (See, also, FIG. 7).Despite this variation, most, if not all, Env polypeptide sequencespreserve the virus's ability to bind to the viral receptor CD4. Further,alignment of the amino acid sequences of Env polypeptide of any HIVvariant can be determined relative to other variants, such as HXB-2, asdescribed for example, in WO 00/39303. In other embodiments, the Envpolypeptide comprises an oligomeric form of Env, for example oligomericgp140 (o-gp140).

The Env polypeptide component of the Env-CD4 complex can be derived anyknown HIV isolates, as well as newly identified isolates, and subtypesof these isolates. Descriptions of structural features can be givenherein with reference to SF2 or HXB-2. One of ordinary skill in the artin view of the teachings of the present disclosure and the art candetermine corresponding regions in other HIV variants (e.g., isolatesHIV_(IIIb), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV), HIV_(LAI),HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strains from diversesubtypes (e.g., subtypes, A through G, and O), HIV-2 strains and diversesubtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)), and simianimmunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition (W. K.Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D.M. Knipe, eds. 1991); Virology, 3rd Edition (Fields, B N, D M Knipe, P MHowley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.; for adescription of these and other related viruses), using for example,sequence comparison programs (e.g., BLAST and others described herein)or identification and alignment of structural features (e.g., a programsuch as the “ALB” program described herein that can identify n-sheetregions). The actual amino acid sequences of the modified Envpolypeptides can be based on any HIV variant.

The Env polypeptides described herein may include additionalmodifications to the native sequence, such as additional internaldeletions, additions and substitutions. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through naturally occurring mutational events. Thus, forexample, if the Env polypeptide is to be used in vaccine compositions,the modifications must be such that immunological activity (i.e., theability to elicit an antibody response to the polypeptide) is not lost.Similarly, if the polypeptides are to be used for diagnostic purposes,such capability must be retained. The Env polypeptides described hereincan be monomeric or oligomeric.

CD4 Mini-Proteins

In the practice of the present invention, Env polypeptides are complexedto CD proteins in order to change conformation of the Env polypeptideand expose epitopes that elicit neutralizing antibodies.

The amino acid sequence of CD4 is known and structural studies on CD4have shown that this molecule is composed of four extracellularimmunoglobulin like domains (three containing disulfide linked loops).It is also known that the binding of gp120 to its receptor (CD4) inducesconformational changes in the Env protein. However only domain 1 (D1) ofCD4 is critical for its interaction with gp120 (Arthos et al. (1989)Cell 57(3):469-81; Truneh et al. (1991) J Biol Chem 266(9):5942-8).Mutational analyses, antibody competition experiments combined with theknowledge of three-dimensional structure of CD4 have shown that a regionhomologous to complementarity determining region 2 (CDR2) ofimmunoglobulin in D1 plays a major role in gp120 binding (Ryu et al.(1994) Structure 2(1):59-74, Sullivan et al. (1998) J Virol72(8):6332-8). Indeed, structure resolution of gp120:CD4 complexconfirmed that the CDR2-like loop of CD4 is central in CD4-gp120interaction (Choe & Sodroski (1992) J Acquir Immune Defic Syndr5(2):204-10, Gizachew et al. (1998) Biochemistry 37(30):10616-25). Inthe complex CD4 Phe-43 side chain plugs the entrance of a deep cavity ingp120 and CD4 Arg59, just behind Phe43, is involved in a double H-bondwith Asp-368 in gp120.

Crystallographic structure analysis of gp120, in complex with CD4 andthe Fab portion the neutralizing monoclonal antibody 17b (Kwong et al.(1998) Nature 393:648-659), indicates that a large surface (742 Å2) ofthe domain D1 of CD4 binds to a large depression (800 Å2) on gp120. TheCD4 interface is comprised by 22 residues, contributing to gp120 bindingwith mixed hydrophobic, electrostatic, H-bonding interactions. The largesize and complexity of this interface makes the reproduction of suchfunctional epitope into a small molecule a challenge, and explains thedifficulty in the development of small molecule inhibitors of gp120-CD4interaction. Vita et al. (1998) Biopolymers 47:93-100. However, in spiteof the large number of residues present in gp120-CD4 interactionsurface, studies on hormone-receptor systems showed that only a fewresidues might dominate the binding energy at the protein-proteininterface. Clackson and Wells (1995) Science 267(5196):383-386.

Upon binding of gp120 to CD4, unique neutralizing epitopes also appearto be exposed, for example the epitope recognized by the monoclonalantibody CG10 (Gershoni et al. (1993) Faseb J7(12):1185-7). Indeed,while monomeric gp120 protein from lab strains is poorly immunogenicwith regard to eliciting primary isolate neutralizing antibodies(Mascola et al. (1996) J. Infect. Dis. 173:340-348), monoclonalantibodies that appear to recognize certain epitopes that are exposed onthe Env surface once it binds to its CD4 receptor have been shown toneutralize diverse primary isolates. See, e.g., the antibody designated17b (Thali et al. (1993) J Virol 67(7):3978-88). However, cross-cladeprimary isolate neutralizing antibody responses usingreceptor/co-receptor complexed Env have been attributed to theimmunogenicity of the gp41 fusion domain. Lacasse et al (1999) Science283:357-362.

Additionally, attempts to evaluate gp120-CD4 complexes as potentialvaccine candidate for inducing high avidity and primary isolateneutralizing antibodies have been thwarted by the concern that an immuneresponse could be generated against CD4 itself thereby raisingautoimmune and safety issues. (D'Souza et al. (1997) J Infect Dis.175:1056-62, DeVico et al. (1995) Virology 211(2):583-8). Since thestructure of CD4 was solved, attempts were made by several groups toidentify minimum gp120 binding domain of CD4 that can retain gp120binding activity (118).

Thus, the present invention makes use of less than full-length CD4proteins or CD4 mimics complexed to an Env protein. Preferably, the CDminiproteins complexed to Env comprises sequences are derived from orfunctionally mimic the D1 domain of CD4. In preferred embodiments, theCD4 mini-protein is derived from, and/or exhibits structural similarityto, the CDR2-like loop of CD4. Non-limiting examples of CD4 miniproteins includes SEQ ID NO:1-4 (Example 4). Structural similarity canbe determined as described herein.

(Vita et al. (1999) Proc Natl Acad Sci USA 96(23):13091-6 and Martin etal. (2003) Nat. Biotech. 21:71-76) describe an additional non-limitingexample of a polypeptide having sequence and/or structural homology toCDR2-like loop of CD4 is the scorpion toxin scyllatoxin (31 residuesonly) that contains a solvent exposed β-hairpin. When its backbone atomsare superimposed on the CDR2-like loop of CD4, (sequence 36-47), an RMSdeviation of only 1.1 Å is found.

One of skill in the art can readily determine amino acid sequences thatexhibit structural and/or amino acid similarity to the D1 domain of CD4in view of the teachings herein. Further, any of these homologs(structural or sequence), can be further modified. Such modificationscan affect structure and/or function. For example, amino acidsubstitutions, additions and/or deletions can be made to themini-proteins such that the gp120 binding structure is preserved orenhanced.

Additional modifications, for example to destroy unwanted functions, canalso be made. For example, as described in Vita et al. (1999) Proc NatlAcad Sci USA 96(23):13091-6 and Martin et al. (2003) Nat. Biotech.21:71-76, a mimetic was generated from scorpion scyllatoxin thatpreserved the structurally important Cys residues of the scaffold, and,additionally, included the solvent-exposed Gly38, Gln40 to Phe43, Thr45and Gly47 of CD4 in structurally equivalent regions of the scyllatoxinβ-hairpin. To further increase the structural mimicry with the CD4, anArg and a Lys were included at positions 7 and 18, topologicallyequivalent to the functional Arg59 and Lys35 of CD4, respectively. Todestroy the original K⁺ channel binding function of scyllatoxin, Arg6and Arg13 were mutated into Ala and Lys, respectively. Finally tworesidues both at the N- and C-terminus were deleted (Vita et al. (1999)Proc Natl Acad Sci USA 96(23):13091-6; Martin et al. (2003) Nat.Biotech. 21:71-76).

Any of the CD4 mini-proteins useful in the practice of the invention canbe chemically synthesized. Preferably, the synthesis is conducted underconditions that allow and promote efficient folding of the mini-proteininto a conformation that binds gp120 and exposes epitopes in or near theCD4 binding site. For example, the mini-protein can be synthesized underconditions that produce a circular dichroism spectrum similar to that ofscyllatoxin, in spite of mutations in the native sequence.

Hybrid Env-CD4 Proteins

In certain aspects, the Env-CD4 molecules are hybrid proteins. In oneembodiment, the hybrids have at least one CD4 protein (e.g., CD4mini-protein) inserted into an Env sequence. In other embodiments, theCD4 sequence(s) precede or follow the Env polypeptide (e.g., by at the Nor C terminal of the Env polypeptide). Further, any of the hybrids mayinclude one or more linkers of varying length (e.g., 3-30 amino acids inlength, or any integer therebetween). In certain embodiments, theEnv-CD4 hybrids are fusion proteins encoded by one or morepolynucleotide sequences.

Suitable CD4 sequences for use Env-CD4 hybrids are described above. Asnoted above, CD4 miniproteins complexed to Env comprises sequences arepreferably derived from or functionally mimic the D1 domain of CD4. Inpreferred embodiments, the CD4 mini-protein is derived from, and/orexhibits structural similarity to, the CDR2-like loop of CD4.Non-limiting examples of CD4 mini proteins includes SEQ ID NO:1-4(Example 4) or CD4 mimetics (such as those described in Vita et al.(1999) Proc Natl Acad Sci USA 96(23):13091-6 and Martin et al. (2003)Nat. Biotech. 21:71-76).

The CD4 mini-protein can be of any length, for example between about 10and about 200 amino acids in length (or any integer therebetween),preferably between about 20 and about 100 amino acids in length, andmore preferably between about 25 and about 85 amino acids in length (orany integer value therebetween). One of skill in the art can readilydetermine amino acid sequences that exhibit structural and/or amino acidsimilarity to the D1 domain of CD4 (and/or SEQ ID NO:1-4) in view of theteachings herein. Further, any of these homologs (structural orsequence), can be further modified.

Typically, the CD4 polypeptide is inserted into any Env polypeptidedescribed herein, for example, gp120, gp140, o-gp140, gp160 or fragmentsthereof. In addition, the Env sequence can contain modifications, forexample, deletions, truncations and/or substitutions. In certainembodiments, one or more of the variable regions (V1 though V5) aredeleted and/or truncated. For example, the CD4 sequence can be insertedinto one of these variable regions. It is preferable, although notrequired, that the CD4 sequence be a single, contiguous sequence. Incertain embodiments, the deleted variable regions are replaced with ashorter polypeptide sequences (e.g., 3-20 amino acid sequence or anyinteger therebetween), as these shorter polypeptides may maintain of theoverall conformation of the Env protein and/or provide the flexibilityneeded to allow for binding of the CD4 protein to Env (Example 4).

As will be readily apparently, one or more elements of the Env-CD4hybrids may contain further modifications. Such modifications can affectstructure and/or function. For example, amino acid substitutions,additions and/or deletions can be made to the mini-proteins such thatthe gp120 binding structure is preserved or enhanced.

Any of the hybrid Env-CD4 proteins useful in the practice of theinvention can be chemically synthesized. Preferably, the synthesis isconducted under conditions that allow and promote efficient folding ofthe mini-protein into a conformation that binds gp120 and exposesepitopes in or near the CD4 binding site. Further, as described indetail below, hybrid Env-CD4 proteins can be produced recombinantly.

Polypeptide Production

The CD4 mini-proteins, Env polypeptides and hybrid Env-CD4 polypeptidesof the present invention can be produced in any number of ways all ofwhich are well known in the art.

In one embodiment, the polypeptides are generated using recombinanttechniques, well known in the art. In this regard, oligonucleotideprobes can be devised based on the known sequences of the Env (e.g.,gp120) polypeptide genome and used to probe genomic or cDNA librariesfor Env genes. The gene can then be further isolated using standardtechniques and, e.g., restriction enzymes employed to truncate the geneat desired portions of the full-length sequence. Similarly, the Envgene(s) can be isolated directly from cells and tissues containing thesame, using known techniques, such as phenol extraction and the sequencefurther manipulated to produce the desired truncations. See, e.g.,Sambrook et al., supra, for a description of techniques used to obtainand isolate DNA.

The genes encoding the modified or hybrid (e.g., truncated and/orsubstituted) Env and/or CD4 polypeptides can be produced synthetically,based on the known sequences. The nucleotide sequence can be designedwith the appropriate codons for the particular amino acid sequencedesired. The complete sequence is generally assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambairet al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311; Stemmer et al. (1995) Gene 164:49-53.

Recombinant techniques are readily used to clone a gene encoding an Envpolypeptide gene that can then be mutagenized in vitro by thereplacement of the appropriate base pair(s) to result in the codon forthe desired amino acid. Such a change can include as little as one basepair, effecting a change in a single amino acid, or can encompassseveral base pair changes. Alternatively, the mutations can be effectedusing a mismatched primer that hybridizes to the parent nucleotidesequence (generally cDNA corresponding to the RNA sequence), at atemperature below the melting temperature of the mismatched duplex. Theprimer can be made specific by keeping primer length and basecomposition within relatively narrow limits and by keeping the mutantbase centrally located. See, e.g., Innis et al, (1990) PCR Applications:Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol.(1983) 100:468. Primer extension is effected using DNA polymerase, theproduct cloned and clones containing the mutated DNA, derived bysegregation of the primer extended strand, selected. Selection can beaccomplished using the mutant primer as a hybridization probe. Thetechnique is also applicable for generating multiple point mutations.See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982)79:6409.

Once coding sequences for the desired Env and/or CD4 proteins have beenisolated or synthesized, they can be cloned into any suitable vector orreplicon for expression. As will be apparent from the teachings herein,a wide variety of vectors encoding modified polypeptides can begenerated by creating expression constructs which operably link, invarious combinations, polynucleotides encoding Env and/or CD4polypeptides having deletions or mutation therein. Thus, polynucleotidesencoding a particular Env having modified (e.g., deleted and/orsubstituted) variable regions (e.g., V1N2) can be operably linked withpolynucleotides encoding polypeptides having deletions or replacementsin the small loop region and the construct introduced into a host cellfor polypeptide expression. Non-limiting examples of such combinationsare discussed in the Examples.

Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra;Sambrook et al., supra; B. Perbal, supra.

Insect cell expression systems, such as baculovirus systems, can also beused and are known to those of skill in the art and described in, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, interalia, Invitrogen, San Diego Calif. (“MaxBac” kit).

Plant expression systems can also be used to produce the modified Envproteins. Generally, such systems use virus-based vectors to transfectplant cells with heterologous genes. For a description of such systemssee, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; and Hackland etal., Arch. Virol. (1994) 139:1-22.

Viral systems, such as a vaccinia based infection/transfection system,as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby etal., J. Gen. Virol. (1993) 74:1103-1113, will also find use with thepresent invention. In this system, cells are first transfected in vitrowith a vaccinia virus recombinant that encodes the bacteriophage T7 RNApolymerase. This polymerase displays exquisite specificity in that itonly transcribes templates bearing T7 promoters. Following infection,cells are transfected with the DNA of interest, driven by a T7 promoter.The polymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA that is thentranslated into protein by the host translational machinery. The methodprovides for high level, transient, cytoplasmic production of largequantities of RNA and its translation product(s).

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired polypeptide is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. With the present invention, both thenaturally occurring signal peptides or heterologous sequences can beused. Leader sequences can be removed by the host in post-translationalprocessing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.Such sequences include, but are not limited to, the TPA leader, as wellas the honey bee mellitin signal sequence.

Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Such regulatory sequences are known to those of skillin the art, and examples include those which cause the expression of agene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Other typesof regulatory elements may also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector. Alternatively, thecoding sequence can be cloned directly into an expression vector thatalready contains the control sequences and an appropriate restrictionsite.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. Mutants oranalogs may be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are well knownto those skilled in the art. See, e.g., Sambrook et al., supra; DNACloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Vero293 cells, as well as others. Similarly, bacterial hosts such as E.coli, Bacillus subtilis, and Streptococcus spp., will find use with thepresent expression constructs. Yeast hosts useful in the presentinvention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by growing host cells transformed byan expression vector described above under conditions whereby theprotein of interest is expressed. The selection of the appropriategrowth conditions is within the skill of the art.

In one embodiment, the transformed cells secrete the polypeptide productinto the surrounding media. Certain regulatory sequences can be includedin the vector to enhance secretion of the protein product, for exampleusing a tissue plasminogen activator (TPA) leader sequence, aninterferon (γ or α) signal sequence or other signal peptide sequencesfrom known secretory proteins. The secreted polypeptide product can thenbe isolated by various techniques described herein, for example, usingstandard purification techniques such as but not limited to,hydroxyapatite resins, column chromatography, ion-exchangechromatography, size-exclusion chromatography, electrophoresis, HPLC,immunoadsorbent techniques, affinity chromatography,immunoprecipitation, and the like.

Alternatively, the transformed cells are disrupted, using chemical,physical or mechanical means, which lyse the cells yet keep the Envpolypeptides substantially intact. Intracellular proteins can also beobtained by removing components from the cell wall or membrane, e.g., bythe use of detergents or organic solvents, such that leakage of the Envpolypeptides occurs. Such methods are known to those of skill in the artand are described in, e.g., Protein Purification Applications: APractical Approach, (E. L. V. Harris and S. Angal, Eds., 1990).

For example, methods of disrupting cells for use with the presentinvention include but are not limited to: sonication or ultrasonication;agitation; liquid or solid extrusion; heat treatment; freeze-thaw;desiccation; explosive decompression; osmotic shock; treatment withlytic enzymes including proteases such as trypsin, neuraminidase andlysozyme; alkali treatment; and the use of detergents and solvents suchas bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. Theparticular technique used to disrupt the cells is largely a matter ofchoice and will depend on the cell type in which the polypeptide isexpressed, culture conditions and any pre-treatment used.

Following disruption of the cells, cellular debris is removed, generallyby centrifugation, and the intracellularly produced Env polypeptides arefurther purified, using standard purification techniques such as but notlimited to, column chromatography, ion-exchange chromatography,size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbenttechniques, affinity chromatography, immunoprecipitation, and the like.

For example, one method for obtaining the intracellular Env polypeptidesof the present invention involves affinity purification, such as byimmunoaffinity chromatography using anti-Env specific antibodies, or bylectin affinity chromatography. Particularly preferred lectin resins arethose that recognize mannose moieties such as but not limited to resinsderived from Galanthus nivalis agglutinin (GNA), Lens culinarisagglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pealectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinumagglutinin (AUA). The choice of a suitable affinity resin is within theskill in the art. After affinity purification, the polypeptides can befurther purified using conventional techniques well known in the art,such as by any of the techniques described above.

Relatively small polypeptides, i.e., up to about 50-100 amino acids inlength, can be conveniently synthesized chemically, for example by anyof several techniques that are known to those skilled in the peptideart. In general, these methods employ the sequential addition of one ormore amino acids to a growing peptide chain. Normally, either the aminoor carboxyl group of the first amino acid is protected by a suitableprotecting group. The protected or derivatized amino acid can then beeither attached to an inert solid support or utilized in solution byadding the next amino acid in the sequence having the complementary(amino or carboxyl) group suitably protected, under conditions thatallow for the formation of an amide linkage. The protecting group isthen removed from the newly added amino acid residue and the next aminoacid (suitably protected) is then added, and so forth. After the desiredamino acids have been linked in the proper sequence, any remainingprotecting groups (and any solid support, if solid phase synthesistechniques are used) are removed sequentially or concurrently, to renderthe final polypeptide. By simple modification of this general procedure,it is possible to add more than one amino acid at a time to a growingchain, for example, by coupling (under conditions which do not racemizechiral centers) a protected tripeptide with a properly protecteddipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M.Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce ChemicalCo., Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, ThePeptides Analysis, Synthesis, Biology, editors E. Gross and J.Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, forsolid phase peptide synthesis techniques; and M. Bodansky, Principles ofPeptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J.Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1,for classical solution synthesis.

Typical protecting groups include t-butyloxycarbonyl (Boc),9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl,acetyl, o-nitrophenylsulfonyl and the like.

Typical solid supports are cross-linked polymeric supports. These caninclude divinylbenzene cross-linked-styrene-based polymers, for example,divinylbenzene-hydroxymethylstyrene copolymers,divinylbenzene-chloromethylstyrene copolymers anddivinylbenzene-benzhydrylaminopolystyrene copolymers.

The polypeptide analogs of the present invention can also be chemically,prepared by other methods such as by the method of simultaneous multiplepeptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985)82:5131-5135; U.S. Pat. No. 4,631,211.

Env-CD4 Complexes

Env and CD4 proteins can be produced as hybrids (e.g., fusion) proteinsas described herein. In addition, Env and CD4 proteins can be separatelyproduced and complexed to each other in a variety of ways. In certainembodiments, Env and CD4 proteins are complexed using one or morecross-linking agents, such as formaldehyde, glutyraldehyde and the like.An alternative strategy will be to link CD4 miniprotein to the envelopeby a specific covalent bond which will not perturb the envelope exposedantigenic surface, yet it will expose the cryptic conserved epitopesthat are normally not accessible, for example so that an antibodyresponse can be mounted. In still further embodiments, a fixative suchas formalin is used to complex Env and CD4 proteins.

In addition, suitable complexes may be produced by e.g., co-transfectinghost cells with constructs encoding hybrid Env-CD4 proteins, Env (e.g.,gp120), CD4 mini-proteins and/or other polypeptides of the desiredcomplex. Co-transfection can be accomplished either in trans or cis,i.e., by using separate vectors or by using a single vector that bearsboth of the Env and the CD4 mini-protein(s). If done using a singlevector, both genes can be driven by a single set of control elements. Asingle set of control elements is preferably employed in the case ofconstructs encoding Env-CD4 hybrid proteins (see, e.g., Example 4).Alternatively, the Env- and CD4 mini protein-encoding genes can bepresent on the vector in individual expression cassettes, driven byindividual control elements. Following expression, the proteins mayspontaneously associate. Alternatively, the complexes can be formed bymixing the individual proteins together which have been producedseparately, either in purified or semi-purified form, or even by mixingculture media in which host cells expressing the proteins, have beencultured. See, International Publication No. WO 96/04301, published Feb.15, 1996, for a description of such complexes.

Antibodies

Antibodies, both monoclonal and polyclonal, which are directed againstEnv-CD4 mini protein complexes epitopes (and cryptic epitopes exposed bybinding of CD4 to Env) are particularly useful in diagnosis andtherapeutic applications, for example, those antibodies which areneutralizing are useful in passive immunotherapy. Monoclonal antibodies,in particular, may be used to raise anti-idiotype antibodies.

Anti-idiotype antibodies are immunoglobulins that carry an “internalimage” of the antigen of the infectious agent against which protectionis desired. Techniques for raising anti-idiotype antibodies are known inthe art. See, e.g., Grzych (1985), Nature 316:74; MacNamara et al.(1984), Science 226:1325, Uytdehaag et al (1985), J. Immunol. 134:1225.These anti-idiotype antibodies may also be useful for treatment and/ordiagnosis of HIV.

An immunoassay for viral antigen may use, for example, a monoclonalantibody directed towards a viral epitope, a combination of monoclonalantibodies directed towards epitopes of one viral polypeptide,monoclonal antibodies directed towards epitopes of different viralpolypeptides, polyclonal antibodies directed towards the same viralantigen, polyclonal antibodies directed towards different viral antigensor a combination of monoclonal and polyclonal antibodies.

Immunoassay protocols may be based, for example, upon competition, ordirect reaction, or sandwich type assays. Protocols may also, forexample, use solid supports, or may be by immunoprecipitation. Mostassays involve the use of labeled antibody or polypeptide. The labelsmay be, for example, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays that amplify the signals from the probe are alsoknown. Examples of which are assays that utilize biotin and avidin, andenzyme-labeled and mediated immunoassays, such as ELISA assays.

An enzyme-linked immunosorbent assay (ELISA) can be used to measureeither antigen or antibody concentrations. This method depends uponconjugation of an enzyme to either an antigen or an antibody, and usesthe bound enzyme activity as a quantitative label. To measure antibody,the known antigen is fixed to a solid phase (e.g., a microplate orplastic cup), incubated with test serum dilutions, washed, incubatedwith anti-immunoglobulin labeled with an enzyme, and washed again.Enzymes suitable for labeling are known in the art, and include, forexample, horseradish peroxidase. Enzyme activity bound to the solidphase is measured by adding the specific substrate, and determiningproduct formation or substrate utilization colorimetrically. The enzymeactivity bound is a direct function of the amount of antibody bound.

To measure antigen, a known specific antibody is fixed to the solidphase, the test material containing antigen is added, after anincubation the solid phase is washed, and a second enzyme-labeledantibody is added. After washing, substrate is added, and enzymeactivity is estimated colorimetrically, and related to antigenconcentration.

Polyclonal antibodies can be produced by administering the fusionprotein to a mammal, such as a mouse, a rabbit, a goat, or a horse.Serum from the immunized animal is collected and the antibodies arepurified from the plasma by, for example, precipitation with ammoniumsulfate, followed by chromatography, preferably affinity,chromatography. Techniques for producing and processing polyclonalantisera are known in the art.

Monoclonal antibodies directed against epitopes exposed by binding ofCD4 to Env (e.g., cryptic epitopes) can also be produced. Normal B cellsfrom a mammal, such as a mouse, immunized with, e.g., an Env-CD4 complexas described herein can be fused with, for example, HAT-sensitive mousemyeloma cells to produce hybridomas. Hybridomas producing antibodiesspecific for epitopes exposed when CD4 miniproteins bind to Env can beidentified using RIA or ELISA and isolated by cloning in semi-solid agaror by limiting dilution. Clones producing the desired specificantibodies are isolated by another round of screening.

Antibodies, either monoclonal and polyclonal, which are directed againstepitopes, are particularly useful for detecting the presence of antigensin a sample, such as a serum sample from an HIV-infected human. Animmunoassay for an HIV antigen may utilize one antibody or severalantibodies. An immunoassay for an HIV antigen may use, for example, amonoclonal antibody directed towards an HIV epitope, a combination ofmonoclonal antibodies directed towards epitopes of one Env or Env-CD4polypeptide, monoclonal antibodies directed towards epitopes ofdifferent polypeptides, polyclonal antibodies directed towards the sameHIV antigen, polyclonal antibodies directed towards different HIVantigens, or a combination of monoclonal and polyclonal antibodies.Immunoassay protocols may be based, for example, upon competition,direct reaction, or sandwich type assays using, for example, labeledantibody. The labels may be, for example, fluorescent, chemiluminescent,or radioactive.

The polyclonal or monoclonal antibodies may further be used to isolateEnv or CD4 complexed-Env by immunoaffinity columns. The antibodies canbe affixed to a solid support by, for example, adsorption or by covalentlinkage so that the antibodies retain their immunoselective activity.Optionally, spacer groups may be included so that the antigen bindingsite of the antibody remains accessible. The immobilized antibodies canthen be used to bind the target from a biological sample, such as bloodor plasma. The bound proteins or complexes are recovered from the columnmatrix by, for example, a change in pH.

In still further aspects, any of the antibodies generated as describedherein (e.g., monoclonal antibodies) can be used to identify epitopes inEnv that may be involved in virus neutralization (e.g., cryptic)epitopes. For example, an epitope recognized by an antibody generated asdescribed herein can be identified and then used to generate furtherantibodies (e.g., neutralizing antibodies). Methods of identifyingepitopes recognized by antibodies are known to those of skill in theart.

Diagnostic, Vaccine and Therapeutic Applications

The Env-CD4 hybrids and/or complexes of the present invention (or thepolynucleotides coding therefor), can be used for a number of diagnosticand therapeutic purposes. For example, the proteins and polynucleotidesor antibodies generated against the same, can be used in a variety ofassays, to determine the presence of reactive antibodies/and or Envproteins in a biological sample to aid in the diagnosis of HIV infectionor disease status or as measure of response to immunization.

As noted above, the presence of antibodies reactive with the Env (e.g.,gp120) polypeptides and, conversely, antigens reactive with antibodiesgenerated thereto, can be detected using standard electrophoretic andimmunodiagnostic techniques, including immunoassays such as competition,direct reaction, or sandwich type assays. Such assays include, but arenot limited to, western blots; agglutination tests; enzyme-labeled andmediated immunoassays, such as ELISAs; biotin/avidin type assays;radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. Thereactions generally include revealing labels such as fluorescent,chemiluminescent, radioactive, or enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between theantigen and the antibody or antibodies reacted therewith.

Solid supports can be used in the assays such as nitrocellulose, inmembrane or microtiter well form; polyvinylchloride, in sheets ormicrotiter wells; polystyrene latex, in beads or microtiter plates;polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, and the like.

Typically, the solid support is first reacted with the biological sample(or the gp120 proteins), washed and then the antibodies, (or a samplesuspected of containing antibodies), applied. After washing to removeany non-bound ligand, a secondary binder moiety is added under suitablebinding conditions, such that the secondary binder is capable ofassociating selectively with the bound ligand. The presence of thesecondary binder can then be detected using techniques well known in theart. Typically, the secondary binder will comprise an antibody directedagainst the antibody ligands. A number of anti-human immunoglobulin (Ig)molecules are known in the art (e.g., commercially available goatanti-human Ig or rabbit anti-human Ig). Ig molecules for use herein willpreferably be of the IgG or IgA type, however, IgM may also beappropriate in some instances. The Ig molecules can be readilyconjugated to a detectable enzyme label, such as horseradish peroxidase,glucose oxidase, Beta-galactosidase, alkaline phosphatase and urease,among others, using methods known to those of skill in the art. Anappropriate enzyme substrate is then used to generate a detectablesignal.

Alternatively, a “two antibody sandwich” assay can be used to detect theproteins of the present invention. In this technique, the solid supportis reacted first with one or more of the antibodies directed against Env(e.g., gp120), washed and then exposed to the test sample. Antibodiesare again added and the reaction visualized using either a direct colorreaction or using a labeled second antibody, such as ananti-immunoglobulin labeled with horseradish peroxidase, alkalinephosphatase or urease.

Assays can also be conducted in solution, such that the viral proteinsand antibodies thereto form complexes under precipitating conditions.The precipitated complexes can then be separated from the test sample,for example, by centrifugation. The reaction mixture can be analyzed todetermine the presence or absence of antibody-antigen complexes usingany of a number of standard methods, such as those immunodiagnosticmethods described above.

The Env-CD4 molecules, produced as described above, or antibodies to thecomplexes, can be provided in kits, with suitable instructions and othernecessary reagents, in order to conduct immunoassays as described above.The kit can also contain, depending on the particular immunoassay used,suitable labels and other packaged reagents and materials (i.e. washbuffers and the like). Standard immunoassays, such as those describedabove, can be conducted using these kits.

The Env-CD4 complexes and/or hybrids (and polynucleotides encoding thecomponent polypeptides) can also be used in vaccine compositions,individually or in combination, in e.g., prophylactic (i.e., to preventinfection) or therapeutic (to treat HIV following infection) vaccines.The vaccines can comprise mixtures of one or more of the complexesand/or hybrids (or nucleotide sequences encoding these molecules), suchas Env (e.g., gp120) proteins derived from more than one viral isolate.The vaccine may also be administered in conjunction with other antigensand immunoregulatory agents, for example, immunoglobulins, cytokines,lymphokines, and chemokines, including but not limited to IL-2, modifiedIL-2 (cys125-ser125), GM-CSF, IL-12, alpha- or gamma-interferon, IP-10,MIT1 and RANTES. The vaccines may be administered as polypeptides or,alternatively, as naked nucleic acid vaccines (e.g., DNA), using viralvectors (e.g., retroviral vectors, adenoviral vectors, adeno-associatedviral vectors, alphaviral vectors) or non-viral vectors (e.g.,liposomes, particles coated with nucleic acid or protein). The vaccinesmay also comprise a mixture of protein and nucleic acid, which in turnmay be delivered using the same or different vehicles. The vaccine maybe given more than once (e.g., a “prime” administration followed by oneor more “boosts”) to achieve the desired effects. The same compositioncan be administered as the prime and as the one or more boosts.Alternatively, different compositions can be used for priming andboosting.

The vaccines will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such vehicles.

A carrier is optionally present which is a molecule that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Furthermore, the Env polypeptide may be conjugated to abacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.

Adjuvants may also be used to enhance the effectiveness of the vaccines.Such adjuvants include, but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents such as muramyl peptides (seebelow) or bacterial cell wall components), such as for example (a) MF59(International Publication No. WO 90/14837), containing 5% Squalene,0.5% Tween 80, and 0.5% Span 85 (optionally containing various amountsof MTP-PE (see below), although not required) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below)either microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponinadjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) detoxified mutants of a bacterialADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (wherelysine is substituted for the wild-type amino acid at position 63)LT-R72 (where arginine is substituted for the wild-type amino acid atposition 72), CT-S109 (where serine is substituted for the wild-typeamino acid at position 109), and PT-K9/G129 (where lysine is substitutedfor the wild-type amino acid at position 9 and glycine substituted atposition 129) (see, e.g., International Publication Nos. W093/13202 andW092/19265); and (7) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Microparticles are also useful as adjuvants. These are preferablyderived from a poly(α-hydroxy acid), in particular, from a poly(lactide)(“PLA”), a copolymer of D,L-lactide and glycolide or glycolic acid, suchas a poly(D,L-lactide-co-glycolide) (“PLG” or “PLGA”), or a copolymer ofD,L-lactide and caprolactone. The microparticles may be derived from anyof various polymeric starting materials that have a variety of molecularweights and, in the case of the copolymers such as PLG, a variety oflactide:glycolide ratios, the selection of which will be largely amatter of choice, depending in part on the coadministered antigen.

The molecules (hybrids, complexes (or polynucleotides encoding the same)and/or adjuvants) may be entrapped within the microparticles, or may beadsorbed to them. Entrapment within PLG microparticles is preferred. PLGmicroparticles are discussed in further detail in Morris et al., (1994),Vaccine, 12:5-11, in chapter 13 of Mucosal Vaccines, eds. Kiyono et al.,Academic Press 1996 (ISBN 012410587), and in chapters 16 & 18 of Vaccinedesign: the subunit and adjuvant approach, eds. Powell & Newman, PlenumPress 1995 (ISBN 0-306-44867-X).

LT mutants may advantageously be used in combination withmicroparticle-entrapped antigen, resulting in significantly enhancedimmune responses.

Typically, the vaccine compositions are prepared as injectables, eitheras liquid solutions or suspensions; solid forms suitable for solutionin, or suspension in, liquid vehicles prior to injection may also beprepared. The preparation also may be emulsified or encapsulated inliposomes for enhanced adjuvant effect, as discussed above.

The vaccines will comprise a therapeutically effective amount of theEnv-CD4 molecules (complexes and/or hybrids) or nucleotide sequencesencoding the same, antibodies directed to these molecules and any otherof the above-mentioned components, as needed. By “therapeuticallyeffective amount” is meant an amount that will induce a protectiveimmunological response in the uninfected, infected or unexposedindividual to whom it is administered. Such a response will generallyresult in the development in the subject of a secretory, cellular and/orantibody-mediated immune response to the vaccine. Usually, such aresponse includes but is not limited to one or more of the followingeffects; the production of antibodies from any of the immunologicalclasses, such as immunoglobulins A, D, E, G or M; the proliferation of Band T lymphocytes; the provision of activation, growth anddifferentiation signals to immunological cells; expansion of helper Tcell, suppressor T cell, and/or cytotoxic T cell.

Preferably, the effective amount is sufficient to bring about treatmentor prevention of disease symptoms. The exact amount necessary will varydepending on the subject being treated; the age and general condition ofthe individual to be treated; the capacity of the individual's immunesystem to synthesize antibodies; the degree of protection desired; theseverity of the condition being treated; the particular Env-CD4 complexselected and its mode of administration, among other factors. Anappropriate effective amount can be readily determined by one of skillin the art. A “therapeutically effective amount” will fall in arelatively broad range that can be determined through routine trials.

Once formulated, the nucleic acid vaccines may be accomplished with orwithout viral vectors, as described above, by injection using either aconventional syringe or a gene gun, such as the Accell® gene deliverysystem (PowderJect Technologies, Inc., Oxford, England). Delivery of DNAinto cells of the epidermis is particularly preferred as this mode ofadministration provides access to skin-associated lymphoid cells andprovides for a transient presence of DNA in the recipient. Both nucleicacids and/or peptides can be injected or otherwise administered eithersubcutaneously, epidermally, intradermally, intramucosally such asnasally, rectally and vaginally, intraperitoneally, intravenously,orally or intramuscularly. Other modes of administration include oraland pulmonary administration, suppositories, needle-less injection,transcutaneous and transdermal applications. Dosage treatment may be asingle dose schedule or a multiple dose schedule. Administration ofnucleic acids may also be combined with administration of peptides orother substances.

Polynucleotide Delivery

As noted above, polynucleotide sequences coding for the above-describedmolecules (hybrids and/or complexes) can be obtained using recombinantmethods, such as by screening cDNA and genomic libraries from cellsexpressing the gene, or by deriving the gene from a vector known toinclude the same. Furthermore, the desired gene can be isolated directlyfrom cells and tissues containing the same, using standard techniques,such as phenol extraction and PCR of cDNA or genomic DNA. See, e.g.,Sambrook et al., supra, for a description of techniques used to obtainand isolate DNA. The gene of interest can also be producedsynthetically, rather than cloned. The nucleotide sequence can bedesigned with the appropriate codons for the particular amino acidsequence desired. In general, one will select preferred codons for theintended host in which the sequence will be expressed. The completesequence is assembled from overlapping oligonucleotides prepared bystandard methods and assembled into a complete coding sequence. See,e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984)223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311; Stemmer, W. P. C.,(1995) Gene 164:49-53.

Next, the gene sequence encoding the desired can be inserted into avector. Insertions can be made within the coding sequence or at eitherend of the coding sequence. Vectors may include control elementsoperably linked to the coding sequence, which allow for the expressionof the gene in vivo in the subject species. For example, typicalpromoters for mammalian cell expression include the SV40 early promoter,a CMV promoter such as the CMV immediate early promoter, the mousemammary tumor virus LTR promoter, the adenovirus major late promoter (AdMLP), and the herpes simplex virus promoter, among others. Othernonviral promoters, such as a promoter derived from the murinemetallothionein gene, will also find use for mammalian expression.Typically, transcription termination and polyadenylation sequences willalso be present, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

The constructs may be uni-cistronic or, alternatively, multi-cistroniccassettes (e.g., bi-cistronic cassettes) can be constructed allowingexpression of multiple antigens from a single mRNA using the EMCV IRES,or the like.

Once complete, the constructs are used for nucleic acid immunizationusing standard gene delivery protocols. Methods for gene delivery areknown in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859,5,589,466. Genes can be delivered either directly to the vertebratesubject or, alternatively, delivered ex vivo, to cells derived from thesubject and the cells reimplanted in the subject.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. Selected sequences can be insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human GeneTherapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnset al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrieand Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett etal., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human GeneTherapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barret al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering the polynucleotides of thepresent invention is the enterically administered recombinant poxvirusvaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

Additional viral vectors which will find use for delivering the nucleicacid molecules encoding the antigens of interest include those derivedfrom the pox family of viruses, including vaccinia virus and avianpoxvirus. By way of example, vaccinia virus recombinants expressing thegenes can be constructed as follows. The DNA is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the codingsequences of interest into the viral genome. The resultingTK-recombinant can be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the genes. Recombinant avipox viruses,expressing immunogens from mammalian pathogens, are known to conferprotective immunity when administered to non-avian species. The use ofan avipox vector is particularly desirable in human and other mammalianspecies since members of the avipox genus can only productivelyreplicate in susceptible avian species and therefore are not infectivein mammalian cells. Methods for producing recombinant avipoxviruses areknown in the art and employ genetic recombination, as described abovewith respect to the production of vaccinia viruses. See, e.g., WO91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis, Semliki Forest, and Venezuelan EquineEncephalitis viruses, will also find use as viral vectors for deliveringthe polynucleotides of the present invention. For a description ofSindbis-virus derived vectors useful for the practice of the instantmethods, see, Dubensky et al., J. Virol. (1996) 70:508-519; andInternational Publication Nos. WO 95/07995 and WO 96/17072; as well as,Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1,1998, and Dubensky, Jr., T. W., U. S. Pat. No. 5,789,245, issued Aug. 4,1998, both herein incorporated by reference.

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient expression of the coding sequencesof interest in a host cell. In this system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into protein by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virusrecombinants, or to the delivery of genes using other viral vectors, anamplification system can be used that will lead to high level expressionfollowing introduction into host cells. Specifically, a T7 RNApolymerase promoter preceding the coding region for T7 RNA polymerasecan be engineered. Translation of RNA derived from this template willgenerate T7 RNA polymerase that in turn will transcribe more template.Concomitantly, there will be a cDNA whose expression is under thecontrol of the T7 promoter. Thus, some of the T7 RNA polymerasegenerated from translation of the amplification template RNA will leadto transcription of the desired gene. Because some T7 RNA polymerase isrequired to initiate the amplification, T7 RNA polymerase can beintroduced into cells along with the template(s) to prime thetranscription reaction. The polymerase can be introduced as a protein oron a plasmid encoding the RNA polymerase. For a further discussion of T7systems and their use for transforming cells, see, e.g., InternationalPublication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986)189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al.,Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994)22:2114-2120; and U.S. Pat. No. 5,135,855.

Polynucleotides encoding hybrids and/or complexes as described hereincan also be delivered without a viral vector. For example, the constructcan be packaged in liposomes prior to delivery to the subject or tocells derived therefrom. Lipid encapsulation is generally accomplishedusing liposomes that are able to stably bind or entrap and retainnucleic acid. The ratio of condensed DNA to lipid preparation can varybut will generally be around 1:1 (mg DNA:micromoles lipid), or more oflipid. For a review of the use of liposomes as carriers for delivery ofnucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991)1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp. 512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081);and purified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethyl-ammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be preparedfrom readily available materials using techniques well known in the art.See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198;PCT Publication No. WO 90/11092 for a description of the synthesis ofDOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY(1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA(1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta(1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA(1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szokaand Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; andSchaefer-Ridder et al., Science (1982) 215:166.

The DNA and/or protein(s) can also be delivered in cochleate lipidcompositions similar to those described by Papahadjopoulos et al.,Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

The polynucleotides of interest may also be encapsulated, adsorbed to,or associated with, particulate carriers. Such carriers present multiplecopies of a selected antigen to the immune system and promote trappingand retention of antigens in local lymph nodes. The particles can bephagocytosed by macrophages and can enhance antigen presentation throughcytokine release. Examples of particulate carriers include those derivedfrom polymethyl methacrylate polymers, as well as microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides), known as PLG. See,e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al.,J Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine11(2):149-54, 1993. Suitable microparticles may also be manufactured inthe presence of charged detergents, such as anionic or cationicdetergents, to yield microparticles with a surface having a net negativeor a net positive charge. For example, microparticles manufactured withanionic detergents, such as hexadecyltrimethylammonium bromide (CTAB),i.e. CTAB-PLG microparticles, adsorb negatively charged macromolecules,such as DNA. (see, e.g., Intl Application Number PCT/US99/17308).

Furthermore, other particulate systems and polymers can be used for thein vivo or ex vivo delivery of the gene of interest. For example,polymers such as polylysine, polyarginine, polyornithine, spermine,spermidine, as well as conjugates of these molecules, are useful fortransferring a nucleic acid of interest. Similarly, DEAEdextran-mediated transfection, calcium phosphate precipitation orprecipitation using other insoluble inorganic salts, such as strontiumphosphate, aluminum silicates including bentonite and kaolin, chromicoxide, magnesium silicate, talc, and the like, will find use with thepresent methods. See, e.g., Feigner, P. L., Advanced Drug DeliveryReviews (1990) 5:163-187, for a review of delivery systems useful forgene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No.5,831,005, issued Nov. 3, 1998, herein incorporated by reference) mayalso be used for delivery of a construct of the present invention.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for deliveringpolynucleotides of the present invention. The particles are coated withthe polynucleotide(s) to be delivered and accelerated to high velocity,generally under a reduced atmosphere, using a gun powder discharge froma “gene gun.” For a description of such techniques, and apparatusesuseful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006;5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needle-lessinjection systems can be used (Davis, H. L., et al, Vaccine12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

Recombinant vectors carrying polynucleotides of the present inventionare formulated into compositions for delivery to the vertebrate subject.These compositions may either be prophylactic (to prevent infection) ortherapeutic (to treat disease after infection). The compositions willcomprise a “therapeutically effective amount” of the gene of interestsuch that an amount of the antigen can be produced in vivo so that animmune response is generated in the individual to which it isadministered. The exact amount necessary will vary depending on thesubject being treated; the age and general condition of the subject tobe treated; the capacity of the subject's immune system to synthesizeantibodies; the degree of protection desired; the severity of thecondition being treated; the particular antigen selected and its mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

The polynucleotide compositions will generally include one or more“pharmaceutically acceptable excipients or vehicles” such as water,saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles. Certain facilitators of nucleic acid uptake and/or expressioncan also be included in the compositions or coadministered, such as, butnot limited to, bupivacaine, cardiotoxin and sucrose.

Once formulated, the compositions of the invention can be administereddirectly to the subject (e.g., as described above) or, alternatively,delivered ex vivo, to cells derived from the subject, using methods suchas those described above. For example, methods for the ex vivo deliveryand reimplantation of transformed cells into a subject are known in theart and can include, e.g., dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, lipofectamineand LT-1 mediated transfection, protoplast fusion, electroporation (see,e.g., Draghia et al. (2002) Technol Cancer Res Treat October;1(5):365-72; Heller (2002) Technol Cancer Res Treat October;1(5):317-8), encapsulation of the polynucleotide(s) (with or without thecorresponding antigen) in liposomes, and direct microinjection of theDNA into nuclei.

Direct delivery of compositions described herein in vivo will generallybe accomplished with or without viral vectors, as described above, byinjection using either a conventional syringe or a gene gun, such as theAccell® gene delivery system (PowderJect Technologies, Inc., Oxford,England). The constructs can be injected either subcutaneously,epidermally, intradermally, intramucosally such as nasally, rectally andvaginally, intraperitoneally, intravenously, orally or intramuscularly.Delivery of DNA into cells of the epidermis is particularly preferred asthis mode of administration provides access to skin-associated lymphoidcells and provides for a transient presence of DNA in the recipient.Other modes of administration include oral and pulmonary administration,suppositories, needle-less injection, transcutaneous and transdermalapplications. Dosage treatment may be a single dose schedule or amultiple dose schedule. Administration of nucleic acids may also becombined with administration of peptides or other substances.

While the invention has been described in conjunction with the preferredspecific embodiments thereof, it is to be understood that the foregoingdescription as well as the examples which follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Preparation of GP120-Soluble CD4 (GP120-sCD4) Complexes

Stable purified gp120-sCD4 complexes were prepared with and withoutformaldehyde or formalin treatment. To induce conformational changes inthe gp120, equimolar concentration of gp120 (SF2) and sCD4 wereincubated together at 37° C. for one hour. At the cellular level, theseinteractions are transient. Therefore, at the end of incubation, half ofthe complexes were fixed with formaldehyde or formalin while the otherhalf remained untreated. Both the treated and untreated complexes wereseparated on Superdex-200 column. Purified fractions were analyzed on anHPLC column and on SDS-PAGE (FIG. 4, panels A and B). The purifiedcomplexes contained both gp120 and CD4 together. Furthermore, thesecomplexes appeared to be homogeneous and did not contain more than 2-3%of free sCD4.

Example 2 Rational Design OF A CD4 Mini-Protein

The CD4 miniprotein, CDM3 was “rationally” designed as described in Vitaet al. (1999) Proc Natl Acad Sci USA 96(23):13091-6, using a structureand function relationship approach. In a first step, thethree-dimensional structure of CD4 miniprotein was determined by ¹H-NMRspectroscopy. This analysis showed that the miniprotein contained theα/β fold characteristic of the scorpion scaffold, and importantly, thatthe putative active site, transferred from CD4, was very well defined.The backbone atoms of the sequence 17-26 of CD4M3 could be superimposedon the corresponding atoms of the sequence 37-46 of native CD4 with aRMS deviation of 0.61 Å only. Furthermore, the side chains of G1n20,Ser22, Phe23 and Thr25 had an orientation very similar to that of thecorresponding side chains in CD4. In particular, the Phe23 side chainwas very well defined because of many long-range contacts observed. Thisside chain protruded into the solvent in a conformation that is ratherunusual for a hydrophobic moiety, but is reminiscent of that of Phe43 ofCD4, which, in the crystal structure of the CD4-gp120 complex, is seento plug the entrance of the “Phe43 cavity” of gp120 (Kwong et al. (1998)Nature 393:648-659). Lys16, Arg7 side chains and Gly27, however,diverged from the structure of the corresponding Lys35, Arg59 and strandC″ of CD4.

In a second step, each putatively active side chain of the miniproteinwas replaced by an Ala residue (“Ala scanning”). The effect of alaninesubstitution on gp120 binding, determined by competitive ELISA, clearlyindicated that each transferred residue played a different functionalrole, and pointed to a Phe residue present at the apex of the β-hairpin,as a “hot spot” of the chimera active surface. This is in agreement withthe data obtained on mutagenesis of recombinant CD4 (Arthos et al.(1989) Cell 57(3):469-81; Binley et al. (1997) AIDS Res Hum Retroviruses13:1007-15). Interestingly, this analysis suggested two substitutions,Gln20Ala and Thr25Ala.

Once these mutations were introduced into the miniprotein (CD4M8, FIG.2), it increased its binding affinity for gp120 by more than one orderof magnitude (Vita (1999) Proc Natl Acad Sci USA 96(23):13091-6). Threemore mutations, Leu18Lys, Ser9Arg, Pro28 were suggested by thestructural analysis and when these mutations were incorporated, thebinding affinity was increased further by ten-fold as compared toprevious double mutant (CD4M8). These 5 mutations produced an optimizedmini-CD4 (CD4M9, FIG. 2) that improved binding to gp120 with an IC50 of400 nM. CDM33 is a 27 amino acid mimic of CD4 and is described in Martinet al. (2003) Nature Biotech. 21:71-76.

Example 3 Additional Rationally Designed Mini CD4 Proteins

As described in Vita et al. (1999) Proc Natl Acad Sci USA 96(23):13091-6and Martin et al. (2003) Nat. Biotech. 21:71-76, further rationallydesigned mini CD4 proteins are made by introducing an azidophoto-reactive function on the distal (para) position of the Phe23phenyl ring, which, by analogy with sCD4 Phe43, should block theentrance of the gp120 hydrophobic “Phe43 cavity.” The azidophoto-reactive function can be easily produced chemically from thecommercially available p-amino-phenylalanine and, upon irradiation at250 nm, forms a reactive nitrene moiety that undergoes fast insertionreaction in electron-rich amino acid side chains (e.g., aromatic sidechains), which are indeed numerous in the hydrophobic “Phe43 cavity.”Alternatively, a haloacetamide group, reactive to methionine orhistidine residues, present close to the “Phe43 cavity,” is incorporatedon the p-amino-phenylalanine 23 or on another position at the interface.Because of the strategic position of the Phe23 side chain, penetratingthe deep “Phe43 cavity” of gp120, the photo- or chemical reactionresults in a relatively homogeneous and high yields covalent complex.

Example 4 Engineering and Biological Expression of CD4Miniprotein-Envelope Fusion Proteins

Polynucleotides encoding the following CD4 mini protein sequences arecloned into an expression construct comprising a sequence encoding anHIV Env polypeptide (e.g., a sequence encoding a modified gp120-encodingpolynucleotide as described, for instance in International PublicationsWO 00/39302 and WO 03/020876):

(SEQ ID NO: 1) gggggTCTASQQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNIRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLggggg (SEQ ID NO: 2)gggggQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNIRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEggggg (SEQ ID NO:3)gggggNSNQIKILGNQGSFLTKGPSKLNIRADSRRSLWDQGNggggg  (SEQ ID NO:4)gggggGNQGSFLTKGPSKLNIRADSRRSLWDQGNggggg

In these sequences, residues marked by underlining are the actualresidues that are in contact with gp120 and residues shown in lower case(“g” for glycine) are linker residues that provide flexibility togp120-CD4 for making a complex.

Any of SEQ ID NO:1-4 are inserted in place of V1, V2, V3, V4 and/or V5loops of a Env-encoding sequences, for example the gp120-encodingsequence shown in FIG. 7 (SEQ ID NO:5). Thus, the insertion can be madeinto any one of the V-loops and, in addition, the construct may have oneor more additional loops deleted. V1 and V2, in particular arerelatively close to the CD4 binding site and can be deleted in the coreprotein in the oligomeric gp140_(SF162), without loss of stability andbinding function. Kwong et al., supra. Shorter insertions (e.g.,corresponding to the CDR2-like loop only) may also be also inserted inplace of one or more variable regions. Models of the Env-CD4 hybrids areproduced by using the CD4M9-gp120_(HXB2) model structure used tooptimize CD4 miniprotein binding affinity (see above) and deduced fromthe CD4-gp120_(HXB2) structure. Different linker sequences are tested(e.g., to minimize energy). The hybrids (chimeras) with the lowestenergies will be further analyzed and compared with the non-covalentcomplex structures. Chimeras with the simple insertion of CD4 CDR2-likeregion will be treated similarly.

In addition, a 27 amino acid CD4 mini protein as described in Martin etal. (2003) Nat Biotechnol January:21(1):71-6 is inserted into the V1 orV2 loops. The stability and folding efficiency of the mimetic scaffoldsuggest that, if sufficient flexibility is allowed at the V-loopinsertion sites, the CD4 miniprotein will fold properly, once insertedinto the protein envelope.

Expression is followed by standard techniques. Further, the binding ofCD4 protein to Env is monitored by the induction of CD4 inducibleepitopes recognized by MAbs 17b and 4.8d using standard techniques. Inparticular, purified chimeras produced in the SF162 Env background,then, are characterized by SPR to evaluate exposure of CD41 epitopes andby co-receptor binding tests to evaluate their binding affinities. Incompetitive ELISA, a rationally designed miniprotein was able tospecifically bind gp120 at an IC50 of 40 uM, which are four orders ofmagnitude higher than that of sCD4. See, also Devico et al. (1999)Virology 218:258-263 and Zhang et al. (1999) Biochemistry 38(29):9405-16which show surface plasmon resonance (SPR) testing of another CD4miniprotein and that the CD4 miniproteins are able to compete with sCD4for binding to the same gp120 site, and to induce envelopeconformational changes, as detected by the monoclonal antibody 17b(Sullivan et al. (1998) J Virol 72(8):6332-6338). This antibodyrecognizes an epitope located near the gp120 V3 loop and consistingmainly of the conserved stem of V1/V2, which is probably masked by theflanking V1N2 and V3 loops (Kwong et al. (1998) Nature (London)393:648-659; Rizzuto et al. (1998) Science 280:1949-1953) but exposed inthe gp120 complexed to CD4. The effect of miniprotein addition onantibody maximum binding and association rate increase was small,probably reflecting its low gp120 binding affinity, but specific andeasily detected.

In sum, these results demonstrate that i) a significant portion of gp120binding surface of CD4 can be reproduced in a miniprotein system, andii) the engineered CD4 mimic and/or CD4-Env hybrids contain enough CD4structural elements able to induce gp120 conformational changes, similarto those expressed by sCD4.

Electroporation and other methods described herein are used toefficiently deliver polynucleotides encoding the fusion proteins tonon-human primates. The DNA prime/protein boost strategy allows forscreening of multiple Env structures in rabbits and non-human primateswith the potential for epitope presentation in situ in the host whendelivered as DNA vaccines.

Example 5 Neutralizing Antibody Production Using CD4-Env Complexes

A. Env-sCD4 Complexes

Rabbit sera were tested in neutralizing assays both against the T-celladapted (TCLA) as well as primary HIV-1 strains. Antibodies induced inthese rabbits by the gp120-sCD4 complexes were able to neutralize boththe SF162 primary isolate as well as T-cell adapted isolates, SF2(homologous strain) and RF (heterologous). To demonstrate thatanti-gp120 antibodies were responsible for neutralizing the virus, anSF2 gp120 affinity column was used to purify the Env-specific antibodyfractions from these sera. The majority of anti-gp120 specificantibodies (95%) bound to the gp120 affinity columns. These were elutedwith 200 mom Glycine pH 2.5. Anti-CD4 antibodies did not efficientlybind to the affinity column. Affinity purified anti-gp120 antibodieswere further evaluated for their anti-gp120 and anti-CD4 reactivities inELISA as well as in an HPLC based'assay. Using this strategy,approximately 90% of the anti-gp120 antibodies were affinity purified,however column eluted gp120 specific antibody fractions were slightlycontaminated with antibodies specific to CD4 (FIG. 5). Accordingly,affinity purified anti-gp120 specific antibodies were further purifiedby passage over a CD4 affinity column to absorb anti-CD4 antibodies.After absorption, anti-gp120 affinity purified antibodies were free fromany detectable anti-CD4 antibodies. These affinity purified anti-gp120antibodies were able to neutralize both subtype B and C primary HIV-1isolates (FIG. 6 and Table 1, below). The values shown in Table 1represent the reciprocal of the highest dilution at which 50% virusinhibition was observed in a PBMC-based virus neutralization assay (byDr. Carl Wild at Panics Corporation, Gaithersburg, Md.).

TABLE 1 Neutralizing activity of gp120 column fractions against HIV-1subtype B and C primary isolates HIV-1_(12298P) HIV-1_(3899S) ElutionFraction (Subtype B) (Subtype C) 1 25 62 2 26 39 3 32 <20B. Rationally-Designed mini CD4 Protein-Env Complexes

Two rabbits each were immunized with fixed or unfixed gp120-sCD4complexes (prepared as described in Example 1) in MF59 at 0, 4, 12 and24 weeks. Sera were collected biweekly and analyzed against SF2 gp120 inan ELISA. These animals mounted a strong immune response against gp120(FIG. 3A). In general, fixed (e.g., formalin-fixed) complexes were moreimmunogenic as reflected by high antibody titers obtained in this groupcompared to the group that received unfixed complexes. In addition toanti-gp120 responses, these complexes also induced strong anti-CD4response, as expected (FIG. 3B)

Thus, the rationally designed CD4 miniprotein, bound with high affinityto different envelope forms (including oligomeric and monomeric forms ofSF162 with and without V2-deletes), induced conformational changes inthese proteins as efficiently as sCD4, and induced full exposition ofconserved cryptic CD4 inducible epitopes and/or co-receptor bindingsites. Thus, the optimized CD4 miniprotein appears to represent a fullyfunctional substitute of sCD4 and that engineering further CD4miniproteins may result in surrogate molecules that may be useful incomplex with envelope protein to expose envelope epitopes toneutralizing antibodies thus may find potential application in vaccineformulations.

C. Env-CD4 Hybrids Proteins

Two rabbits each are immunized with constructs encoding Env-CD4 hybridsat 0, 4, 12 and 24 weeks. Sera are collected biweekly and analyzed in anELISA. Env-CD4 hybrid proteins (and polynucleotides encoding thesehybrids) are expected to represent a fully functional substitute of sCD4and may be useful in expose envelope epitopes to neutralizing antibodiesthus may find potential application in vaccine formulations.

D. Monkeys

Groups of 5 rabbits are immunized with Env-CD4 miniprotein complexes orEnv-CD4 hybrid proteins with adjuvant (MF59) along with control groupsof Env protein only and CD4 miniprotein only. CD4 complexes and Env-CD4hybrids are made with monomeric and oligomeric forms of SF162 Env withand without V2-deletes and the antibody responses in rabbits compared.Immunization schedules are at 0, 4, and 24 week immunizations; whenwarranted, an additional booster may be included at 24 weeks. Env-CD4complexes identified by these rabbit studies are then tested inmacaques.

Example 6 Unmasking Cryptic Epitopes Of GP41 Subunit in OligomericEnvelopes

CD4 miniprotein induces a conformational transformation of oligomeric(o-gp140) envelopes, unmasking cryptic epitopes, close to co-receptorsites in gp120 subunit and efficiently increases co-receptor bindingaffinity in different gp120 envelopes. Whether this conformationaltransformation can also expose epitopes within the gp41 subunit ofo-gp140 envelopes has not been tested. Accordingly, the induction ofthis conformational transformation by CD4 miniproteins binding in thedifferent oligomeric Env structures is tested, using SPR technology and2F5 mAb or DP178 peptides (or congeners). The effect of addition ofpeptides from the N-terminal domain of CCR5 co-receptor, which have beenshown to bind to gp120 is also examined.

If exposition of gp41 epitopes is demonstrated, the peptides arechemically coupled to the CD4 miniprotein, to produce novelbi-functional ligands, presenting increased potency in unmasking gp41epitopes. Novel chimeric oligomeric envelopes, incorporating thebi-functional ligands are also produced chemically or genetically, andtested. Candidate envelope proteins with superior exposure of gp120 andgp41 cryptic epitopes are then tested in animals for the induction ofneutralizing antibodies.

Example 7 Production of Monoclonal Antibodies Targeting CrypticConserved Epitopes of Env

Selected Env-CD4 immunogens will be injected in rats to preparemonoclonal antibodies, according to the standard procedures. Clones willbe screened in ELISA against CD4 miniprotein-gp120 complex, CD4miniprotein-o-gp140, gp120 and o-gp140 alone and CD4M33 miniprotein aswell. All the clones exhibiting highest affinity for complexes ascompared to envelopes alone will be further tested in Biacore. All theclones scoring positive in Biacore against the CD4M33-gp120 and orCD4M33-o-gp140 complexes will be selected and used for bulk productionof ascites fluids.

1. A hybrid Human Immunodeficiency Virus (HIV) Envelope (Env)-CD4protein comprising: (1) an HIV Env polypeptide which comprises aCD4-binding site and a deletion region from which one or more variable(V) regions are deleted; and (2) a CD4 mini-protein or a CD4-mimeticinserted into the deletion region, wherein the CD4 mini-protein orCD4-mimetic maintains the structural conformation of a CDR2-like loop;and wherein the insertion of the CD4 mini-protein or the CD4 mimeticleads to exposure of a cryptic HIV envelope epitope in or near theCD4-binding site or in or near the chemokine receptor-binding site. 2.The polypeptide of claim 1, wherein the deletion region comprises adeletion in V1.
 3. The polypeptide of claim 1, wherein the deletionregion comprises a deletion in V2.
 4. The polypeptide of claim 1,wherein the deletion region comprises a deletion in V1 and V2.
 5. Thepolypeptide of claim 1, further comprising one or more linker sequences.6. The polypeptide of claim 1 wherein the one or more linker sequencesflank the CD4 mini-protein or the CD4 mimetic.
 7. The polypeptide ofclaim 1, wherein the CD4 mini-protein is selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. 8.The polypeptide of claim 1 wherein the CD4 mini-protein is inserted inthe deletion region.
 9. The polypeptide of claim 1 wherein the CD4mimetic is inserted in the deletion region.
 10. The polypeptide of claim1 wherein the HIV Env polypeptide comprises gp140.
 11. A compositioncomprising the polypeptide of claim
 1. 12. The composition of claim 11,further comprising an adjuvant.
 13. A method of inducing an immuneresponse in a subject, comprising administering to the subject thecomposition of claim
 11. 14. The method of claim 13 wherein the subjectis a mammal.
 15. The method of claim 14 wherein the subject is a human.